Dermal micro-organs, methods and apparatuses for producing and using the same

ABSTRACT

Embodiments of the present invention provide Dermal Micro-organs (DMOs), methods and apparatuses for harvesting the same. Some embodiments of the invention provide a DMO including a plurality of dermal components, which substantially retain the micro-architecture and three dimensional structure of the dermal tissue from which they are derived. An apparatus for harvesting the DMO may include, according to some exemplary embodiments, a support configuration to support a skin-related tissue structure from which the DMO is to be harvested, and a cutting tool able to separate the DMO from the skin-related tissue structure. Exemplary embodiments of the invention provide a genetically modified dermal micro-organ expressing at least one recombinant gene product. Some embodiments of the invention provide methods and apparatuses for implanting a genetically modified DMO.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/686,939, filed Nov. 28, 2012, which is a Continuation in Part of U.S.application Ser. No. 13/369,329, filed Feb. 9, 2012, which is aDivisional Application of U.S. application Ser. No. 12/216,321 now U.S.Pat. No. 8,142,990, filed Jul. 2, 2008, which is a Continuation of U.S.application Ser. No. 10/834,345, filed Apr. 29, 2004, now U.S. Pat. No.7,468,242, which claims priority from U.S. Provisional Application No.60/466,793, filed May 1, 2003, and U.S. Provisional Application No.60/492,754, filed Aug. 6, 2003 and is a Continuation in Part of PCTInternational Application Numbers PCT/IL02/00877, PCT/IL02/00878,PCT/IL02/00879 and PCT/IL02/00880, all filed Nov. 5, 2002, thedisclosures of all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The invention relates to the field of tissue based micro-organs,therapeutic tissue based micro-organs and methods and apparatuses forharvesting, processing, implanting and manipulating dermal tissue.

BACKGROUND OF THE INVENTION

Various methods for delivering therapeutic agents are known. Forexample, therapeutic agents can be delivered orally, transdermally, byinhalation, by injection and by depot with slow release. In each ofthese cases the method of delivery is limited by the body processes thatthe agent is subjected to, by the requirement for frequentadministration, and limitations on the size of molecules that can beutilized. For some of the methods, the amount of therapeutic agentvaries between administrations.

A dermal micro-organ (DMO), which can be sustained outside the body (“exvivo” or “in vitro”) in an autonomously functional state for an extendedperiod of time, and to which various manipulations can be applied, maythen be implanted subcutaneously or within the body for the purpose oftreating diseases, or disorders, or for plastic surgical purposes. TheDMO can be modified to express a gene product of interest. Thesemodified dermal micro-organs are generally referred to as DermalTherapeutic Micro-Organs (DTMOs).

Skin micro-organs (MO), including layers of epidermal and dermaltissues, for example; as outlined in PCT/IL02/0880, have been observedto be associated with a number of clinical challenges. Harvesting of askin sample leaves a superficial wound on the patient that may lastseveral weeks and may leave scars. The harvested skin sample requiressignificant processing to generate micro-organs from this sample. Also,implantation of skin micro-organs subcutaneously or deeper in the bodyhave been found to result in the development of keratin cysts or keratinmicro-cysts. Additionally, implantation of skin micro-organs as a graftonto the skin surface in “slits” requires significant technicalexpertise in order to handle the MO while maintaining its properorientation.

Harvesting of dermis, e.g., to be used as a “filler material” in aplastic surgical or cosmetic procedure, is known in the art.Conventional harvesting techniques include using a dermatome or scalpelto peel away a layer of epidermis in order to expose a section ofdermis. The dermatome or scalpel may then be used again to manuallyharvest the exposed section of dermis.

Another conventional apparatus for harvesting dermis, albeit notcommonly used, is the Martin Dermal Harvester marketed by Padgett (PartNo. P-225) for the indication of harvesting dermal cores from the backfor subsequent implantation into the lips during cosmetic lipaugmentation procedures. To operate this device, which is not commonlyused, a sharpened cutting tube, which includes a reusable thick walledtube with an inner diameter of approximately 4.5 mm, is manually rotatedat a very slow speed. Using this type of device generally requiresapplying pressure to the skin surface directly above the harvest siteand installing sutures with active tugging as the cutting tube is pushedforward. Furthermore, the resulting harvested dermis is generally notuniform in dimensions and includes “plugs” of epidermis at either end ofthe dermal core.

SUMMARY OF THE INVENTION

Embodiments of some aspects of the present invention provide a DMO/DTMOwith the ability to be maintained ex-vivo in a generally viable state,which may allow various manipulations to be performed on the DMO, whilekeeping a high production and secretion level of the desired therapeuticagent, as disclosed in United States Application Publication No.US-2012/0201793-A1, which is incorporated herein by reference in itsentirety. In addition, embodiments of some aspects of the presentinvention provide a method of harvesting a DMO and subsequentlyimplanting a DTMO without forming keratin cysts or keratin microcysts,e.g., upon implantation of the DTMO subcutaneously or deeper in thebody. Furthermore, it will be appreciated by persons skilled in the artthat the methods and devices according to some embodiments of thepresent invention may be relatively uncomplicated and, therefore, thelevel of skill required from a professional to carry out the methodsand/or to use the devices of the present invention may not be asdemanding as those required in conventional procedures.

Some exemplary embodiments of the invention provide a dermal micro-organ(DMO) having a plurality of dermal components, which may include cellsof the dermal tissue and a surrounding matrix. The DMO according toembodiments of the invention may generally retain a micro-architectureand three dimensional structure of the dermal organ from which it isobtained and the dimensions of the DMO may allow passive diffusion ofadequate nutrients and gases to the cells and diffusion of cellularwaste out of the cells so as to minimize cellular toxicity andconcomitant death due to insufficient nutrition and accumulation ofwaste.

In some exemplary embodiments of the invention, the DMO of the inventiondoes not produce keratin or produces negligible amounts of keratin.

In some embodiments of the invention, the DMO does not produce keratinand/or keratin cysts following subcutaneous or deeper implantation in abody.

In another embodiment of the invention, the DMO of the inventionproduces micro keratin cysts following that will atrophy within arelatively short period of time, e.g., days or weeks after subcutaneousimplantation.

In another embodiment of the invention, the DMO contains hair folliclesand sebaceous glands.

Further, exemplary embodiments of the invention provide a method andapparatus of harvesting a dermal micro-organ. The method may includestabilizing and/or supporting a skin-related tissue structure from whicha dermal micro-organ is to be harvested, e.g., such that theskin-related tissue structure is maintained at a desired shape and/orposition, separating at least a DMO from the skin-related tissuestructure, and isolating the separated DMO from the body. According tosome of these exemplary embodiments, a support structure may include avacuum chamber able to hold the skin-related tissue structure in adesired shape and position to enable a cutting tool to cut a DMO fromthe skin-related tissue structure. In one embodiment the supportstructure includes one or more vacuum channels to fluidically connectthe vacuum chamber with at least one vacuum source.

In one embodiment an apparatus for harvesting a dermal micro-organcomprises (a) a support structure to support a skin-related tissuestructure from which the DMO is to be harvested, the support structurecomprising a first tubular element, and the first tubular elementcomprising a site of insertion into the apparatus; (b) an introducer;and (c) a cutting tool. In some embodiments, the first tubular elementis a guide channel that may guide additional elements, for instance acutting tool, for insertion into the supported skin-related tissue.

In one embodiment, an apparatus of the invention includes a vacuumchamber further comprising: (a.) two elevated protrusions, a near, i.e.,proximal, elevated protrusion and a distal elevated protrusion relativeto the site of insertion, wherein the elevated protrusions are able tosupport a plateau of at least epidermal and dermal skin layers from theskin-related tissue structure above the trajectory of a cutting tool;and (b) a central channel located between the two elevated protrusions,wherein the central channel supports epidermal and dermal skin layersfrom the skin-related tissue structure so that the dermal skin layer iswithin the trajectory of a cutting tool when the cutting tool isinserted in the first tubular element of the apparatus.

In another embodiment, an apparatus of the present invention includes anintroducer comprises a second tubular element and a fourth tubularelement, wherein the second tubular element inserts through the fourthtubular element and extends beyond the distal end of the fourth tubularelement and the second and fourth tubular elements together insert atthe site of insertion coaxially within the first tubular element; andfurther, the fourth tubular element remains coaxial and within the firsttubular element upon withdrawal of the second tubular element.

In yet another embodiment, an apparatus includes a third tubularelement, for instance a cutting tool that inserts within and through thefourth tubular element.

In one embodiment, the cutting tool comprises a coring tube able to cutthrough the skin-related tissue structure when advance along a cuttingaxis, wherein the cutting axis is substantially coaxial with the firsttubular element. In another embodiment, the coring tube is a rotatablecoring tube attached to a power source.

In one embodiment, a vacuum chamber includes a vacuum control mechanism.Implementation of a vacuum condition may for example, include placing afinger over a hole in the vacuum chamber, i.e., a vacuum hole, that whencovered creates a vacuum condition. Alternative, release of a vacuumcondition, may for example include removal of the finger from over thevacuum hole. In alternate embodiments, any covering or uncovering of thevacuum hole may be used to control vacuum and release conditions,respectfully. In one embodiment, a vacuum control mechanism relies onclamping and unclamping the vacuum line or opening and closing a valvein the vacuum control line.

In one embodiment, a method of harvesting a DMO of the inventionincludes the steps of: positioning an apparatus at a harvest site incontact with an epidermal surface of a subject; supporting askin-related tissue structure at the harvest site from which the DMO isto be harvested; puncturing the skin-related tissue structure; cuttingthe DMO from the supported skin-related tissue structure; and recoveringthe DMO. In another embodiment, a harvesting method includes making onlya single puncture point in the skin-related structure.

In one embodiment, a method of harvesting includes the use of a vacuumto recover the DMO from the coring tube into a closed container. In oneembodiment, the closed container is a syringe body. In certaininstances, the syringe may have an attached septum. In anotherembodiment, the DMO remains within the coring tube after retraction fromthe harvest site and recovering the DMO comprises flushing the DMO fromthe coring tube.

Further, exemplary embodiments of the invention provide a method of andapparatus for implanting a dermal micro-organ. In one embodiment, thedermal micro-organ to be implanted is a genetically modified dermalmicro-organ, which may also be referred to herein as a dermaltherapeutic micro-organ (DTMO).

In one embodiment, an apparatus for implanting a DMO or a DTMO includes(a) a loading syringe comprising a first tubular element; (b) animplanting tool comprising a second tubular element; (c) a supportstructure to hold a skin-related tissue structure in place, wherein theDMO is to be implanted within the skin related tissue structure; (d) anintroducer for puncturing the skin at a penetration site; (e) a stoppertool able to be connected to the support structure, the stopper toolcomprising a tubular element, and the stopper tool assisting inmaintaining the position of a DMO during retraction of the implantingtool.

In an exemplarily embodiment of the invention, a method for implanting aDTMO includes the steps of: (a) loading a DTMO into a loading syringe,the loading syringe comprising a first tubular element; (b) transferringthe DTMO from the loading syringe into an implanting tool, theimplanting tool comprising a second tubular element; (c) placing animplanting apparatus at an implantation site, wherein the apparatus isin contact with an epidermal layer of the subject and the implantingaxis is generally parallel with the epidermal layer; (d) supporting askin-related tissue structure at the implantation site wherein the DTMOis to be implanted into the skin-related structure; (e) puncturing theskin within the skin-related tissue structure at the penetration site,wherein the skin is punctured using an introducer including an innerneedle and an outer sleeve element; (f) removing the inner needle of theintroducer and advancing the implanting tool into the skin-relatedtissue structure along the implanting axis; and (g) withdrawing thesecond tubular element wherein the DTMO remains within the skin-relatedtissue structure. In one embodiment, a stopper tool is used to assist inmaintaining the position of a DTMO during retraction of the implantingtool. In certain instances, the first and second steps may be optional,as a DTMO may be loaded directly into the distal end of the implantationneedle by suctioning from the back end of the needle with a syringe.

In another embodiment, a DTMO may be implanted by directly injecting theDTMO from a syringe through a needle whose distal end is positionedunder the skin or at another anatomical location, if linear implantationis not important.

Further exemplary embodiments of the invention provide a geneticallymodified dermal micro-organ expressing at least one recombinant geneproduct the DMO having a plurality of dermal components, including cellsand matrix of the dermal tissue, which retain the micro-architecture andthree dimensional structure of the dermal tissue from which they areobtained, and having dimensions selected so as to allow passivediffusion of adequate nutrients and gases to the cells and diffusion ofcellular waste out of the cells so as to minimize cellular toxicity andconcomitant death due to insufficient nutrition and accumulation ofwaste, wherein at least some of the cells of the DMO express at leastone recombinant gene product or at least a portion of the at least onerecombinant gene product, as described in United States Publication No.US-2012-0201793-A1, and incorporated herein in full. In still otherexemplary embodiments, the at least one recombinant gene product is anat least one recombinant protein.

In some embodiments of the invention, the genetically modified DMO ofthe invention produces substantially no keratin.

In some embodiments, the invention provides a method of delivering to arecipient a recombinant gene product produced by the DMO.

In some embodiments, the invention provides a method of inducing a localor systemic physiological effect by implanting a DMO in a recipient.

In another embodiment the invention provides a method of delivering aprotein of interest to a subject. The method includes implanting thegenetically modified DMO into the skin, under the skin or at otherlocations in the body.

In another embodiment, the invention provides a method of implanting aDTMO so as to avoid or to reduce keratin cyst formation.

In one embodiment, the invention provides a method for removal of animplanted DTMO.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the invention are described in the followingdescription, to be read with reference to the figures attached hereto.In the figures, identical and similar structures, elements or partsthereof that appear in more than one figure are generally labeled withthe same or similar references in the figures in which they appear.Dimensions of components and features shown in the figures are chosenprimarily for convenience and clarity of presentation and are notnecessarily to scale.

FIG. 1 is a schematic block diagram of an exemplary method of producingand utilizing dermal therapeutic micro-organs (DTMOs), in accordancewith an exemplary embodiment of the invention;

FIG. 2 is a schematic flowchart illustrating a method of harvesting aDMO according to some exemplary embodiments of the invention;

FIGS. 3A-3G are schematic illustrations of exemplary stages ofharvesting a DMO in accordance with a method of FIG. 2;

FIGS. 4A-4E show embodiments of some elements of a harvesting apparatus,a medical drill for use with a harvesting apparatus and a syringe,septum and collet for use harvesting the DTMO. 4A shows an embodiment ofa syringe (4002) for harvesting and a septum (4004). 4B shows anembodiment of a collet. 4C shows an embodiment of a support structurewith a vacuum hole being covered by a finger. 4D shows an embodiment ofan introducer: inner needle (4006—needle) and outer guide (4008—whitesleeve). 4E shows an embodiment of a cutting tube (4010), a drill (4012)and a drill hand piece (4014).

FIG. 5A-B are schematic illustration of some components of a dermalharvesting apparatus according to another exemplary embodiment of theinvention. FIG. 5A is a schematic illustration of a lateral view ofembodiments of a harvesting apparatus. FIG. 5B is a schematicillustration of a cross-sectional view of the apparatus of FIG. 5Aexternally supporting a skin-related tissue structure from which adermal micro-organ may be harvested at a desired position;

FIG. 6 is a schematic illustration of some components of a dermalharvesting apparatus according to yet another exemplary embodiment ofthe invention;

FIG. 7 is a flow chart illustrating a DTMO implanting method, accordingto some exemplary embodiments of the present invention;

FIGS. 8A-E show an embodiment of an implanting apparatus. 8A is anembodiment of a loading syringe, 8B is an embodiment of an implantingtool, 8C is an embodiment of an introducer, 8D is an embodiment of asupport structure, and 8E is an embodiment of a stopper.

FIG. 9A-E are schematic illustrations of exemplary stages of implantinga DTMO in accordance with a method of FIG. 7;

FIGS. 10A-B show embodiments of a syringe with a septum and collet. FIG.10A shows a syringe with a septum and collet inserted through a supportstructure guide channel and an outer sleeve, wherein the supportstructure is connected to a vacuum source. FIG. 10B shows an embodimentof a syringe with a collet and needless valve attached to the back endof a coring needle. The T-end of an introducer (1008) is identified fororientation purposes;

FIG. 11 shows a schematic illustration of an embodiment of a supportstructure.

FIG. 12 is a flow chart illustrating a method of removing a previouslyimplanted DTMO;

FIG. 13 shows an embodiment of a syringe (1306) being attached to aglued-on male luercap (1302) near the back end of a coring needle (1304)

FIGS. 14A and 14B show embodiments of a harvested dermal micro-organ(FIG. 14A) and harvesting (1402) and implanting (1404) sites on a humansubject.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity. Further, where consideredappropriate, reference numerals may be repeated among the figures toindicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

The following description is presented to enable one of ordinary skillin the art to make and use the invention as provided in the context of aparticular application and its requirements. Various modifications tothe described embodiments will be apparent to those with skill in theart, and the general principles defined herein may be applied to otherembodiments. Therefore, the present invention is not intended to belimited to the particular embodiments shown and described, but is to beaccorded the widest scope consistent with the principles and novelfeatures herein disclosed. In other instances, well-known methods,procedures, and components have not been described in detail so as notto obscure the present invention. In still other instances, methods,procedures and components described in United States Publication No.US-2012/0201793-A1 are incorporated herein by reference in theirentirety.

I. Exemplary Definitions of Terms Used Herein

The term “explant” as used herein, refers in some embodiments of theinvention, to a removed section of living tissue or organ from one ormore tissues or organs of a subject, wherein an “explant” may forexample be a dermal micro-organ.

The term “dermal micro-organ” or “DMO” as used herein, refers in someembodiments of the invention, to an isolated tissue or organ structurederived from or identical to an explant that has been prepared in amanner conducive to cell viability and function, while maintaining atleast some in vivo interactions similar to the tissues or organ fromwhich it is obtained. DMOs may include plurality of dermal componentsthat retain the micro-architecture of the tissue or organ from whichthey were derived, and three dimensional structure of the dermal tissuefrom which they are derived, having dimensions selected so as to allowpassive diffusion of adequate nutrients and gases to cells within theDMO and diffusion of cellular waste out of the cells of the DMO so as tominimize cellular toxicity and concomitant death due to insufficientnutrition and accumulation of waste. DMOs may consist essentially of aplurality of dermis components (tissue components of the skin locatedbelow the epidermis). These components may contain skin fibroblast,epithelial cells, other cell types, bases of hair follicles, nerveendings, sweat and sebaceous glands, and blood and lymph vessels.Wherever used herein below, the description of the embodiments relatedto DMO relates also to a MO. Further, whenever the term “dermal tissue”is used, it also relates to “dermal organ” and a DMO.

As used herein, the term “microarchitecture” refers, in some embodimentsof the invention, to the characteristic of the explant in which, in oneembodiment at least about 50%, in another embodiment, at least about60%, in another embodiment at least about 70%, in another embodiment, atleast about 80%, and in another embodiment, at least about 90% or moreof the cells of the population, maintain, in vitro, their physicaland/or functional contact with at least one cell or non-cellularsubstance with which they were in physical and/or functional contact invivo. Preferably, the cells of the explant maintain at least onebiological activity of the organ or tissue from which they are isolated.

The term “donor” as used herein, refers in some embodiments of theinvention to a subject, from which the explant is removed and used toform, or which is already in the form of, one or more micro-organs. Inone embodiment, the donor is a human subject. In another embodiment, thedonor is a non-human mammalian subject.

The term “therapeutic micro-organ (TMO)” as used herein, refers in someembodiments of the invention to a dermal micro-organ (DMO) that can beused to facilitate a therapeutic objective, such as, for example, an DMOthat has been genetically altered or modified to produce a therapeuticagent, such as a protein or an RNA molecule. The therapeutic agent mayor may not be a naturally occurring body substance. Wherever usedhereinbelow, the description of the embodiments related to TMO relatesalso to DTMO which is a therapeutic Dermal MO which may be in someembodiments of the invention genetically modified.

The term “implantation” as used herein, refers in some embodiments ofthe invention, to introduction of one or more TMOs or DTMOs into arecipient, wherein said TMOs or DTMOs may be derived from tissues of therecipient or from tissues of another individual or animal. The TMOs orDTMOs can be implanted in a slit within the skin, can be subcutaneouslyimplanted, or may be implanted by placement at other desired siteswithin the recipient body. In one embodiment, a DTMO is derived fromtissue of the recipient. In one embodiment, a DTMO is implantedsubstantially into the dermal layer of skin tissue. In one embodiment, aDTMO is implanted between the dermal and fat layer of skin tissue.

The term “recipient” as used herein refers, in some embodiments of theinvention, to a subject into which one or more TMOs or DTMOs areimplanted. In one embodiment, the recipient is a human subject. Inanother embodiment, the recipient is a non-human mammalian subject. Insome embodiments, a recipient receives one or more autologous TMOs orDTMO.

The term “in vitro” as used herein should be understood to include“ex-vivo”.

The term “coring tube” as used herein may relate, individually orcollectively, to the terms “cutting tool”, “cutting tube” and “coringneedle”, “coring tool”, as well as to any other elements with similarfunctionalities. In some embodiments, a coring needle of this inventionis for single use.

The term “implanting tool” as used herein may relate, individually orcollectively, to the terms “implantation needle”, “implanting needle”and “implanting tube”, as well as to any other elements with similarfunctionalities. In some embodiments, an implanting tool of thisinvention is for single use.

The term “tubular element” as used herein refers to an element havingthe form of or consisting of a tube, wherein a tube refers to any ofvarious usually cylindrical structures or devices. In one embodiment, atubular element is an element having the form of a hollow elongatedcylinder. In another embodiment, a tubular element is an element havingthe form of a cylindrical channel, e.g., a tunnel or channel cut througha solid mass. In yet another embodiment, a tubular element is open atboth ends. In still another embodiment, a tubular element is open at oneend. In a further embodiment, a tubular element comprises a beveledneedle tip at one end. In another embodiment, a tubular elementcomprises at least one blunt end that is sharpened. In one embodiment atubular element is an element having the form of a solid, non-hollowelongated cylinder, for instance a rod. In one embodiment, a tubularelement may include a guide channel.

The term “rod” as used herein refers to a straight three-dimensionalelement, which has a solid geometry. In one embodiment, the rod has acircular cross section. In one embodiment, the rod has a non-circularcross-section.

The term “skin-related tissue structure”, as used herein, refers to astructure of tissue components that may be stabilized and/or supportedby apparatuses defined herein to enable the harvesting of a dermalmicro-organ therefrom or for the implantation of a DMO therein. Askin-related tissue structure may include components of the epidermaltissue, and components of the dermal tissue. Optionally, theskin-related tissue structure may include fat tissue and/or muscletissue in the vicinity of the dermal tissue.

In one embodiment, a skin-related tissue structure of the presentinvention includes the skin tissue components drawn into the centralchannel under vacuum conditions. In one embodiment, a skin-relatedstructure includes epidermal, dermal and fat tissue. In anotherembodiment, a skin-related structure includes epidermal, dermal, fat andmuscle tissue.

The term “central channel” as used herein may in some embodiments of theinvention be used interchangeable with the term “vacuum chamber”.

The term “coaxial”, as used herein, refers to a radial symmetry ofconcentrically or approximately concentrically positioned components. Inthis way, tubular elements may be positions approximately equidistantfrom a common axis. In one embodiment, a cutting tool is alignedapproximately equidistant from a common axis presented by a guidechannel. In one embodiment, a cutting tool is aligned approximatelyequidistant from a common axis presented by a central channel.

As used herein, the term “approximately” refers to a range of valueswithin plus or minus 10% of an ideal. For example, approximately coaxialtubular elements may share the identical central axis or may be havecentral axes that are within 10% of a shared identical central axis.

In one embodiment, one tubular element is contained within anothertubular element, but the central axis of both tubes need not be aligned.

While, for clarity and completeness of presentation, all aspects of theproduction and utilization of DTMOs are described in this document, andembodiments of the invention are described from the start of theprocesses to their ends, it should be understood that each of theaspects described herein can be used with other methodologies and/orequipment for the carrying out of other aspects and can be used forother purposes, some of which are described herein. The presentinvention includes portions devoted to the preparation and maintenanceof dermal micro-organs for transformation into DTMOs. It should beunderstood that the dermal micro-organs produced according to theseaspects of the invention can be used for purposes other than fortransformation into DTMOs

In some embodiments of the invention, the micro-organ is a DMO includinga plurality of dermis components, for example, fibroblasts and/orepithelial components containing nerve endings and/or sweat glandsand/or sebaceous glands and/or blood and lymph vessels and/or elastinfibers and/or collagen fibers and/or endothelial components and/orimmune system derived cells and/or extra-cellular matrix. Conventionalsubcutaneous implantation of a micro-organ including epidermal layers(“split thickness skin MO”) in mice and pigs, may result in formation ofkeratin cysts or macro-keratin cysts. In contrast, when skin tissue issampled to obtain a DMO or when a DMO is directly harvested, accordingto exemplary embodiments of the invention, after subcutaneousimplantation or implantation in other anatomical locations, no cysts ormacro cysts are observed in mice, pigs or in humans. It should be notedthat the biological activity (for example, secretion of a therapeuticprotein, e.g., erythropoietin and elevation of hematocrit as a result)of a DTMO according to embodiments of the invention may be comparable toor even higher than split thickness skin derived TMO.

In general, production of DTMOs may include DMO harvesting, maintainingthe DMO and/or modifying the DMO and/or genetically altering them and,in some embodiments, verifying the production of a desired agent (forexample proteins) by the DMO. Utilization of the DTMO may includeproduction, within a patient's or animal's own body, of therapeuticsubstance, such as proteins, for treatment of a subject. For example,the DTMO can be implanted into or under the skin or within the body ofthe subject to produce the agent/protein in vivo.

In one embodiment, a DTMO is not encapsulated in an immunoprotectivecapsule or sheath.

In some embodiments of the invention, the DMO may contain tissue of abasal epidermal layer and, optionally, other epidermal layers of theskin. In other embodiments, the dermal micro-organ does not includebasal epidermal layer tissue.

In some embodiments of the invention, the DMO does not include epidermallayers. In other embodiments, the DMO may contain a few layers ofepidermal tissue. In some embodiments, the dermal micro-organ may lack acomplete epidermal layer. In certain instances, a DMO may includeinvaginations of the epidermis within the dermal tissue layers, whilestill lacking a complete epidermal layer.

In one embodiment of the invention, the DMO includes the entirecross-section of the dermis. In another embodiment of the invention, thedermal micro-organ includes part of the cross-section of the dermis. Ina further embodiment, the DMO includes most of the cross section of thedermis, namely, most of the layers and components of the dermisincluding the papillary and reticular dermis. In a further embodiment,the DMO includes primarily dermal tissue, but may also include fattissue. In some embodiments of the invention, the DMO does not producekeratin or produces a negligible amount of keratin, thereby preventingthe formation of keratin cysts following implantation in a recipient.

II. Methods and Apparatuses for Harvesting a DMO

The DMO to be harvested can be removed from the body by any means ofremoving tissue known in the art, such as biopsy procedures. Theharvesting procedure keeps intact the micro-architecture of the tissuefrom which it is removed. In one embodiment the DMO may be obtained bydirect biopsy and then be cut to the required size. In anotherembodiment, a tissue sample may be obtained by direct biopsy, in whichthe desired size of the dermal micro-organ is obtained. In anotherembodiment, non-desired tissue may be cut from the harvested biopsy ordirectly harvested micro-organ. In one embodiment, a DMO may be obtainedby direct biopsy and then processed to become a DTMO by geneticmodification of the DMO in vitro. In one embodiment a DMO or a DTMO maybe labeled in vitro for identification purposes, e.g., a DMO or a DTMOmay be colored prior to implantation by an inert, biocompatible ink orstain containing, for example, a chromophore, which may be visible tothe naked eye or may require special illumination conditions tovisualize it.

In some embodiments of the invention, the dermal micro-organ is directlyharvested from the body. In other embodiments, a dermal micro-organ isharvested with the aid of a harvesting apparatus. The inner diameterdimension of a cutting tool used to harvest a dermal micro-organ may be,for example, about 0.5-4 mm. In another embodiment, the dimension maybe, for example, 1.71 mm. In yet another embodiment, the dimension maybe 1.21 mm. In still another embodiment, the dimension may be, forexample, 1-3 mm. In a further embodiment, the dimension may be, forexample, 2-4 mm. In one embodiment the dimension may be, for example,1-2 mm. In another embodiment, the dimension may be 0.5-1.5 mm. In yetanother embodiment, the dimension may be, for example, about 1.5 mm. Instill another embodiment, the dimension may be, for example, about 2 mm.

In some embodiments, the cutting tool has dimensions corresponding toneedle size dimensions. In one embodiment, the cutting tool is, forexample, a 14 GA needle. In another embodiment, the cutting tool is a 15GA needle. In yet another embodiment, the cutting tool is a 16 GAneedle. In yet another embodiment, the cutting tool is a 17 GA needle.In still another embodiment, the cutting tool is an 18 GA needle. In afurther embodiment, the cutting tool is a 19 GA needle. In oneembodiment, the cutting tool is a 12 GA needle. In another embodiment,the cutting tool is a 13 GA needle. The wall thickness of a cutting toolcorresponding to a needle size dimension, may be for example a regularwall thickness (RW), a thin wall thickness (TW), a extra thin wallthickness (XTW), or any thickness known in the art.

The shape of the tip of a cutting tool may also play a role inharvesting a DMO. A sharp tip may be used, as is, e.g., commerciallyavailable needles. Alternatively, a cutting tool may have a tip, whichhas been sharpened, e.g., by polishing or through the use of chemicaltreatments or using electro-chemical erosion. In one embodiment, thesharp tip of a cutting tool is symmetrical sharpened. The sharpening ofthe tip may be either on the OD surface or the ID surface. For example,the tip may be sharpened by removing material from the outer or innersurface of the tip.

In some embodiments, the harvested DMO may not retain its cylindricalshape after harvesting, i.e., at least one dimension of its crosssection may expand while at least another dimension of its cross sectionmay contract. In one embodiment, for example, at least one dimension maybe 0.5-3.5 mm and at least one dimension may be 1.5-10 mm.

In another embodiment, the dimensions of the tissue being harvested maybe, for example, about 5-100 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about10-60 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 20-60 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 20-50 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about20-40 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 20-100 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 30-100 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about40-100 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 50-100 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 60-100 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about70-100 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 80-100 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 90-100 mm in length. In another embodiment the lengthmay be around 20 mm. In another embodiment, the length may be about 30mm. In another embodiment, the length may be about 40 mm.

When a DMO has the above listed dimensions, it may be maintained invitro, e.g., in a growth medium under proper tissue culture conditionsfor extended periods of time, for example, several days, several weeksor several months. The DMO may be maintained, for example, in-vitro indefined growth media. In one exemplary embodiment the growth media mayinclude growth factors, fetal calf serum (FCS), human serum, orSynthetic Serum Substitute (SSS). In another exemplary embodiment thegrowth media may include serum either from the donor or the recipientsubject. In yet another embodiment the growth media may includeautologous serum. In another embodiment, no serum is added to the media.

In accordance with an aspect of some embodiments of the invention, onlya portion of a DTMO generated may be used in a given treatment session.The remaining DTMO tissue may be returned for maintenance and/or may bestored (e.g., cryogenically or otherwise) for later use. In oneembodiment, a DMO is stored, for example cryogenically or otherwise,prior to genetic modification processing. In another embodiment, a DMOis stored, for example cryogenically or otherwise, following geneticmodification processing.

It is a feature of some embodiments of the invention that a large numberof dermal micro-organs may be processed together in a batch process intoDTMOs, as described below. This may allow for more convenientprocessing, but will not allow for determination of the secretion levelof each DTMO separately. In other embodiments, a DMO may be processedindependently into a DTMO, as described herein.

In some exemplary embodiments of the invention a potency assay may beperformed for the therapeutic agent, which may be produced and/orsecreted by either a single DTMO or a batch of DTMOs. The potency assaymay include, for example, a cell proliferation assay in which theproliferation response of the cells is mainly dependent on the presenceof the therapeutic agent in the growth media of the cells. In oneembodiment, analysis of a DTMO may use for example an ELISA assay inorder to quantify secretion levels of an at least one secretedtherapeutic agent.

According to some embodiments of the invention, a method of harvestingthe DMO may include stabilizing and supporting a skin-related tissuestructure from which a DMO is to be harvested, e.g., such that at leastthe DMO and/or one or more other tissue segments in its vicinity aremaintained at a desired shape and/or position, separating at least aportion of the DMO from surrounding tissue, and extracting the separatedDMO, as described in detail below.

FIG. 1 shows an overview of a methodology for producing and utilizingDMOs and DTMOs, in block diagram form, in accordance with an exemplaryembodiment of the invention. Similarly, a DTMO may be produced byfollowing the steps described independent of a bioreactor. At block 202a DMO is harvested from a subject. In some embodiments of the invention,the DMO is harvested from the same subject to which therapy will laterbe applied. In an embodiment of the invention, the DMO is from dermaltissue. Optionally, other tissues are harvested and used in a mannersimilar to that described below with reference to dermal tissue. Whilethe method described below is exemplary, other methods of harvestingtissue samples can be used in some embodiments of the invention. Ifdesired, the DMO or DTMO can be cryogenically stored for later use(i.e., introduction at the same stage of the process). Alternatively,for certain embodiments, the DMO can be implanted directly back into thepatient from which it was harvested to produce a therapeutic, cosmetic,or other physiological affect.

In order for a DMO to be a viable micro-organ, it must have at least onedimension that is small enough that nutrients can diffuse to all thecells of the DMO from a nutrient medium which contacts the DMO and thatwaste products can diffuse out of the DMO and into the medium. Thisenables the DMO to be viable in vitro long enough for the furtherprocessing described below and for the optional further utilization ofthe DMO as a source for a therapeutic agent, such as a protein. Themethod of harvesting a DMO generally results in a DMO having an in vitrolife of several months.

A suitable genetic modification agent is prepared (block 208).Alternative exemplary methods of preparing the agent include creation ofaliquots with a desired amount of a modifying agent using a predefineddilution buffer containing modifying agent, such as for example a viralvector, and validating the activity of the modifying agent. All of theseprocesses are well known in the art. At this point the DMO can be storedcryogenically, for later introduction at the same place in the process.This can be performed using known protocols for gradual freezing oftissues and cells, using for example, DMEM medium containing 10% DMSO

At block 210 the DMO is genetically altered. As described above, manymethods of genetic alteration are known and may be used in conjunctionwith the present invention. As an example, the following description isbased on using a viral vector to insert a gene into the cells of theDMO. This process is well known and will not be further described,except as to the particular methodology and apparatus for introducingthe virus to the DMO.

At block 212 the genetically altered DTMO is optionally tested forproduction and secretion rates of the therapeutic agent. There arevarious methods of determining the quantity of secretion, for example,ELISA, other immunoassays, spectral analysis, etc. In addition thequality of the secretion is optionally tested, for example for sterilityand/or activity of the secreted protein. This may be performedperiodically or continuously on-line. At this point the DTMO can becryogenically stored for later use.

At blocks 214 and 216, the amount of DTMO required for producing adesired therapeutic effect is determined. As indicated below, thetherapeutic dose requirements can be estimated from measured secretionrates, patient parameters and population statistics on the estimated orknown relationship between in vitro secretion and in vivo serum levels.

At block 218 the desired number of the DTMOs are selected. A DTMO isloaded into an implantation tool. Exemplary implementation tools aredescribed below.

If the DTMOs must be transported prior to being transferred to theimplantation tools, it is optionally held (220) in a maintenance stationor under maintenance conditions, in which the temperature, humidity,etc. are held at levels that allow the DTMO to stay viable duringtransport. The remaining DTMOs are optionally maintained in vitro forfuture use. This can be at warm incubator conditions (30-37° C.), inconditions as described above at cool incubator conditions (4° C.),which may prolong its viability in vitro, or under cryogenic conditions.

At block 224, a subset of the DTMOs is implanted into the subject. Anexemplary embodiment of a method of implantation is described below.Other methods of doing so will occur to persons of skill in the art andare primarily dependent on the specific geometry of the micro-organbeing used. Animal studies have shown that the DMOs and DTMOs remainviable in vivo, in the sense that the DTMO continues to produce andsecrete the therapeutic agent for a period of weeks and months followingimplantation. In animal studies, therapeutic amounts are produced forperiods up to 160 days (or longer). While the tissue of the DMO or DTMOappears to be integrated or well taken into the tissue of the subjectinto which it is implanted (especially if the tissue is implanted in atissue of the same kind from which it was harvested), the cells includedwithin the DMO or the DTMO continue to produce and secrete thetherapeutic agent.

The in vivo performance of the DTMO is optionally determined (block228). Based on this evaluation for example, and/or on past patient data(block 226), patient dosage may then be adjusted (block 230) byincreasing the amount of the implant or removing some of the implant, asdescribed below. As the efficacy of the implant changes, additionalDTMOs can be implanted.

Genetic alteration may generally include genetically engineering aselected gene or genes into cells that causes the cells to produce andoptionally to secrete a desired therapeutic agent such as a protein. Inan embodiment of the invention, at least part of the process ofsustaining the DMO during the genetic alteration, as well as the geneticalteration itself, may be performed in a bioreactor.

According to some exemplary embodiments of the invention, a method ofharvesting a DMO from a subject may include generating and/ormaintaining a skin-related tissue structure associated with the DMO,e.g., located generally at a targeted harvest site for harvesting theDMO, at a desired shape and position such that the cutting tool may beable to separate the DMO from tissue in the vicinity of the DMO. Forexample, an epidermis portion in the vicinity of the targeted harvestsite may be lifted, e.g., by attaching at least part of the epidermisportion to a predefined surface such that at least part of theskin-related tissue structure may be lifted and maintained at thedesired shape and/or position. According to some exemplary embodiments,attaching the epidermis to the predefined surface may include applying avacuum condition, e.g., as described below. Alternatively oradditionally, attaching the epidermis to the predefined surface mayinclude applying an adhesive to the surface.

Reference is now made to FIG. 2, which schematically illustrates aflowchart of a method of harvesting a DMO according to some exemplaryembodiments of the invention, and to FIGS. 3A-3G, which schematicallyillustrate exemplary stages of harvesting a DMO 3024 located within askin-related tissue structure.

As indicated at block 2002, the method may optionally include locallyadministering an anesthetic, e.g., as is known in the art, to thevicinity of the DMO to be harvested.

Use of DTMOs for protein or RNA based therapy may, in certaincircumstances, necessitate use of multiple DTMOs. As describedthroughout, DMOs and DTMOs may be maintained in vitro for extended timeperiods or stored cryogenically or otherwise, for later use. Therefore,in some instances, multiple DMOs may be harvested consecutively during asingle procedural time period from the same subject. In this way,multiple DMOs may be harvested from the subject for later use, withoutthe subject undergoing separate harvesting procedures on separate daysfor each DMO needed. In one embodiment, a harvester marker template maybe used prior to positioning a harvester apparatus on a subject'sepidermal surface (step 2004), in order to mark multiple sites forharvesting. In one embodiment, a harvester marker template is positionedon the epidermal surface of a subject, and the epidermal surface is thenmarked to indicate, for example, area for application of localanesthesia, alignment lines and harvesting lines. In one embodiment, thesurface is marked using a surgical pen or marker. In one embodiment, thesurface is marked using a non-permanent dye or ink.

As indicated at block 2004, the method may further include positioningan apparatus including a support structure, (e.g., FIG. 4C; FIG. 5A;FIG. 6; FIG. 11), at a given harvest site so that the support structure,or a portion thereof, is in contact with an epidermal surface of thesubject. In some embodiments, a contact between a support structure ofthis invention and the epidermal surface of a subject must be air-tightso that a vacuum seal may be formed at a later step. In one embodiment,a harvest site is on a subject's back. In another embodiment, a harvestsite is on a subject's abdomen. In yet another embodiment, a harvestsite may be at another location on a subject's body.

As indicated at block 2006 and FIG. 3A, a support structure (e.g., FIG.4C; FIG. 13A; FIG. 6; FIG. 11), which may include a vacuum chamber (FIG.11 1130) and guide channel 2008 (FIG. 11 1108), under vacuum conditionsmay be used to hold and support the skin-related tissue structureincluding epidermal (3000), dermal (3002) and fat (3004) tissue layers,in place for proper harvesting of a DMO. For example, application ofvacuum conditions causes a vacuum to be formed within the vacuum chamberthereby drawing the epidermal surface of the skin-related structure intothe interior of the support structure, wherein a central channel maysupport epidermal and dermal skin layers of the skin-related structure.

As used herein, the term “guide channel” may also be referred to hereinas a “needle guide channel”.

Under vacuum conditions a central channel may provide support for theskin-related tissue structure to be shaped so that dermal tissue iswithin the central channel. In some exemplary embodiments, the vacuumchamber includes one elevated protrusion. In other exemplaryembodiments, the vacuum chamber includes two elevated protrusions. Ininstances where a support structure that includes one or two elevatedprotrusions is used, the elevated protrusions additionally may supportepidermal and dermal skin layers of the skin-related structure.

In certain instances, application of vacuum conditions using a vacuumchamber with two elevated protrusions may create a precise geometry ofthe skin-related structure such that dermal tissue is harvested and aplug of epidermal tissue is not harvested.

In exemplary embodiments, a vacuum condition may cause the skin-relatedstructure to be held at an inner support surface of the vacuum chamber,including within a central channel and elevated protrusions if present.The guide channel 3008, which in one embodiment may be tubular in shape,may provide guidance and/or stability for inserting and/or using acutting tool to ensure proper cutting along a cutting axis. In someembodiments, the cutting axis is coaxial with the guide channel. Whilethe coring tube is coaxial with the guide channel, the coring needle maynot always be in the vertical center of the central channel. In oneembodiment, the coring tube is in the horizontal center of the centralchannel.

In certain embodiments, the inner dimensions of a support structureincluding a vacuum chamber may be between 3.0-8.0 mm. In one embodiment,the dimension may be, for example 3.0 mm in diameter. In anotherembodiment, the dimension may be, for example 3.5 mm in diameter. In ayet another embodiment, the dimension may be, for example 4.0 mm indiameter. In another embodiment, the dimension may be, for example 4.5mm in diameter. In another embodiment, the dimension may be, for example5.0 mm in diameter. In still another embodiment, the dimension may be,for example 5.5 mm in diameter. In yet another embodiment, the dimensionmay be, for example 6.0 mm in diameter. In a further embodiment, thedimension may be, for example 6.5 mm in diameter. In another embodiment,the dimension may be, for example 7.0 mm in diameter. In yet anotherembodiment, the dimension may be, for example 7.5 mm in diameter. Inanother embodiment, the dimension may be, for example 8.0 mm indiameter.

In one embodiment, the appropriate sized support structure havingparticular inner dimensions of a vacuum chamber is pre-determined priorto actual harvesting.

As indicated at block 2008 and FIG. 3B, an introducer (e.g., FIG. 4D),including for instance, an inner needle (3010; 4006) and an outer sleeve(3012; 4008), may then be used to puncture the skin-related tissue byinserting the introducer through the guide channel of the supportstructure, 3006, and into the skin-related tissue structure at a pointof penetration. This single puncture site may be used for all furtherentry and egress into and out of the skin-related structure. In this waydamage and scarring to the subject is limited. Introduction of thecutting tool through the outer sleeve of the introducer prevents orminimizes harvesting of epidermal tissue.

An introducer may be composed of tubular elements, for instance an innerneedle and an outer sleeve. In some embodiments, the outer sleeve isfitted over the inner needle. Together they may be inserted into theskin-related structure. In certain embodiments, where a supportstructure with a vacuum chamber including a proximal elevated protrusionis used the configuration of the skin-related tissue is such thatinsertion of the introducer is generally perpendicular to the skinsurface at the point of penetration. In one embodiment where a supportstructure with a vacuum chamber including a proximal elevated protrusionis used, an introducer may be inserted into the skin-related structuresuch that the tip of the inner needle extends into the region of tissuein the area of the first, i.e., proximal elevated protrusion, 3007, andthe distal end of the outer sleeve extends to about mid-way under thisfirst elevated protrusion. In one embodiment, following insertion of theintroducer, the distal end of the outer sleeve transverses all of thelayers of the skin at the puncture site and is located in the underlyingfat layer.

In the absences of the step at block 2008, using an introducer topuncture the skin, the step at block 2012 below would result in theharvesting of a plug of full thickness skin prior to the harvest of thedermal micro-organ, which would necessitate additional processing of thetissue to remove the full thickness skin plug in order to produce a DMO.

In one embodiment, the inner needle is beveled. In another embodiment,the inner needle is not beveled. In one embodiment, insertion of theintroducer is with the bevel of the inner needle pointed downward. Inanother embodiment, insertion of the introducer is with the bevel of theinner needle pointed upward. In yet another embodiment, insertion of theintroducer is with the bevel of the inner needle at an intermediateangle between upward and downward.

The exemplary embodiment described below (block 2010) is based on theuse of a support structure including, at least, a vacuum chamberincluding proximal (3007) and distal (3009) elevated protrusions.

As indicated at block 2010 and FIG. 3C, the outer sleeve of theintroducer, 3012, is positioned by inserting the introducer through theinner guide channel, through the epidermal 3000 and dermal 3004 layersof the skin-related structure and into the fat layer 3002 located underthe proximal protrusion. Following insertion of the introducer, theinner needle is withdrawn from the skin-related structure and the outersleeve remains in place, such that the distal end of the outer sleeveresides about mid-way under the proximal protrusion. The result of thisaction is that the outer sleeve is positioned coaxially with the innerguide channel, which may also be the cutting axis, and the tip of theouter sleeve extends into the fat (3002) of the skin-related tissuestructure supported by the proximal protrusion 3007.

The outer sleeve, 3012, may be made up of a thin tube, a hollow rod, orany other suitable thin, generally straight, object able to be placedaround the inner needle and able to penetrate the necessary skin layers.For example, in one embodiment, an outer sleeve may have an innerdiameter which corresponds with a needle of size 12-19 GA for example,about 14 GA. In one embodiment, an outer sleeve may include a plastictube of an appropriate length. In one embodiment, the outer sleeveincludes high-density polyethylene (HPDE) tubing. In another embodiment,the outer sleeve includes polytetrafluoroethylene (PTFE) tubing. Inanother embodiment, the outer sleeve includes fluorinated ethylenepropylene (PEP) tubing. In one embodiment the length of the outer sleeveis approximately 10-100 mm. In one embodiment, the length of the outersleeve is approximately 40 mm. In one embodiment, approximately 5-20% ofthe length of the outer sleeve enters the skin-related structure beyondthe puncture site. In one embodiment, approximately 10-15% of the lengthof the outer sleeve enters the skin related structure beyond thepuncture site. In one embodiment, approximately 12.5% of the length ofthe outer sleeve enters the skin-related structure beyond the puncturesite.

The embodiments above describe penetration of an outer sleeve into a fatlayer. In another embodiment, an outer sleeve may be inserted intodermis tissue. In yet another embodiment, an outer sleeve may beinserted into a subcutaneous space.

The length of penetration of the outer sleeve 3012 through the skin intodermis or fat or a subcutaneous space may generally correspond to 1 to15 mm, or in one embodiment about 5 mm. For example, an outer sleeve maybe inserted manually as part of the introducer, and guided to a desireddepth within the dermis, fat or subcutaneous tissue under the proximalprotrusion. The outer sleeve is then co-axial with the cutting axiswithin the central channel, so that dermis may be harvested by thecutting tool.

In one embodiment, use of an outer sleeve protects an entry puncturesite from exposure to the rotational and forward motion of a cuttingtool, thereby preventing additional trauma to the skin at the site ofentry.

As indicated at block 2012 and FIGS. 3D and 3E, a DMO may now be cutfrom the skin-related tissue. A cutting tool, 3014, may be insertedcoaxially through the guide channel 3008 and outer sleeve 3012, suchthat the outer sleeve guides the cutting tool along a cutting axis (FIG.5A and FIG. 6) into the skin-related structure. In one embodiment,guiding a cutting tool through the outer sleeve may allow the cuttingtool to directly enter dermal tissue (3004) of the skin-relatedstructure (3E). As an aide to cutting, the cutting tool may be rotatedas the tube advances towards the distal end of the apparatus. A motormay be used to rotate the cutting tool. A motor may be, for example, apneumatic motor drill or an electric motor drill. In some embodiments, amedical drill may be used to rotate the cutting tool. In one embodiment,the cutting tool attaches at one end to a drill collet (3016), as isknown in the art

In one embodiment the method may include rotating the cutting tool whileadvancing the cutting tool, e.g., towards the distal end of the supportstructure. For example, a medical drill or other suitable tool orrotation mechanism may be used to rotate a coring tube 3014 while it isadvanced manually or automatically, thereby more smoothly cutting dermaltissue for a DMO. For example, a proximal end of coring tube may beconnected to a medical drill using a drill collet, 3016. Examples ofmedical drills include the Aesculap Micro Speed drill manufactured byAesculap AG & Co. KG, Am Aesculap Platz, D-78532 Tuttlingen, Germany,which may include a control unit, a motor, a connection cord, a handpiece and/or a foot switch, catalogue numbers GD650, GD658, GB661, GB166and GB660, respectively; and a Nouvag medical drill, TCM-3000-BL, andhand piece, catalogue numbers 3285 and 1710, respectively. Such a drill,or any other suitable drill or rotation mechanism, may be used to rotatethe cutting edge of the cutting tool at a rotational speed appropriatefor cutting of the dermal tissue, for example, a relatively highrotational speed, for example, a speed higher than 1,000 RPM, e.g.,between 1,000 RPM and 10,000 RPM. For example, tube 3014 may be rotatedat a rotational speed higher than 2,000 RPM, e.g., approximately 7,000RPM. Alternatively, a relatively low rotational speed of less than 1000RPM may be used, or no rotation at all, as described below. Optionally,the rotational speed of the drill may vary in an oscillatory manner,i.e., the direction of rotation may vary periodically between“clockwise” and “counterclockwise” directions. While rotated by a drill,a coring tube may be manually or automatically advanced, e.g., towardsthe distal end of the support structure, e.g., towards the distalelevated protrusion 3009.

In one embodiment, a method of cutting a dermal micro-organ may includestopping the forward motion of the coring tube at a particular location.In one embodiment, the meeting of the collet of the drill (3016) withthe proximal end of the introducer sleeve (3012) at the outer surface ofa support structure (3006) may act as a hard stop, preventing furtherforward motion of a coring tube. In another embodiment, the meeting ofor an element placed on the external distal portion of the coring tube,e.g., a cap encircling the coring tube (FIG. 13; 1302), with the outersurface of a support structure may act as a hard stop, preventingfurther forward motion of a coring tube.

In certain exemplary embodiments, a method of cutting a DMO using asupport structure including a vacuum chamber with at least a distalelevated protrusion may include stopping the forward motion of coringtube, for example, at a position such that the tip of the coring tube(FIG. 3E) has been advanced to reside within the region of theskin-related structure positioned under the distal elevated protrusion3009. In one embodiment, the meeting of the collet of the drill with theintroducer sleeve at the outer surface of a support structure mayposition the distal tip of the coring tube in fat. In one embodiment,the meeting of the collet of the drill with the introducer sleeve at theouter surface of a support structure may position the distal tip of thecoring tube to reside under the distal elevated protrusion such that thetip enters the fat layer.

In one embodiment, the geometry of the skin-related structure createdwithin a vacuum chamber with at least a distal elevated protrusionensures that at termination of forward movement the distal tip of acoring tube will have crossed the dermis/fat interface, so that the tipresides in fat. The dermis/fat interface has a weak connection. In oneembodiment, the weak connection between dermis and fat may ensure thatthe dermal tissue sample can be separated from the body fat duringrecovery of the DMO.

The cutting tool may include any suitable cutting tool, for example, acoring tube (e.g., FIG. 4E; 4010). Coring tube may include a generallysymmetrically sharpened tubular tool, e.g., a hypodermic tube processedto have sharpened cutting edge with a desired shape. A coring tube mayinclude, for example, a standard medical grade tube, having a thin wall,e.g., having a thickness of between 0.05 mm and 0.3 mm. A coring tubemay have an inner diameter, for example, between 0.5 mm-4 mm. In oneembodiment, an inner diameter may be between 1-2 mm. In anotherembodiment, an inner diameter may be between 1-3 mm. In yet anotherembodiment, an inner diameter may be between 2-4 mm. In still anotherembodiment, an inner diameter may be between 0.5-1.5 mm. In oneembodiment, an inner diameter may be about 1.21 mm. In anotherembodiment, an inner diameter may be about 1.5 mm. In still anotherembodiment, an inner diameter may be about 1.71 mm. In yet anotherembodiment, an inner diameter may be about 2 mm. In one embodiment, thecoring tube has about the dimensions of a 14 GA needle. In anotherembodiment, the coring tube has about the dimensions of a 12 GA, 13 GA,15 GA, 16 GA, 17 GA, 18 GA or 19 GA needle. The dimensions, e.g., thediameter, of coring tube and/or the dimensions of introducer may bepredetermined based on the volume and/or dimensions of the DMO intendedto be harvested. A coring tube may have a sharpened end (“tip”) adaptedto serve as a cutting edge. In one embodiment, the sharpened edge issharpened on the outer diameter. In another embodiment, the sharpenededge is sharpened on the inner diameter. A coring tube may be insertedthrough the outer sleeve and into the skin-related tissue structure inorder to prevent harvesting of epidermal tissue. In one embodiment, useof a support structure with at least a proximal elevated protrusioncreates a precise geometry of the skin-related structure when undervacuum conditions such that an epidermal layer and/or a plug ofepidermis is not harvested.

According to some exemplary embodiments of the invention, at least partof an inner surface and/or an outer surface of tube may be coated with alow friction material, e.g., Teflon®, Parylene or any other suitablecoating material, e.g., to ease the separation of the harvested tissuefrom the inner surface of the cutting tool in a subsequent action and/orto reduce any forces acting on the tissue during the cutting action, asdescribed below.

As indicated at block 2014 and FIGS. 3F and 3G, the method of harvestingincludes recovery of the DMO 3024. After a coring tube has been advancedto a hard-stop and the distal tip of the coring tube is located withinthe skin-related tissue structure positioned under the distal elevatedprotrusion (e.g., FIG. 6), the collet of the drill 3016 (FIG. 4H) isopened and while holding the coring tube in place, the drill is removed.In one embodiment, an external cap, for example a luercap, may be use tohold the coring tube in place. Then the collet 3020/septum 3021 of thesyringe 3022 assembly is attached (FIGS. 4B, 4A (4004) and 4 (4012),respectively) to the coring tube.

In one embodiment, a male luercap (1302) is attached, e.g., glued, tothe outer surface of a coring needle (1304). The ability to hold theglued cap in place while removing the drill and connecting the syringe,prevents movement of the coring needle during the final steps ofharvesting of a DMO. This may prevent potential loss of a DMO.

First the collet 3020 is attached. This is a stand-alone collet, notthat of the medical drill. The collet may be slipped over the coringtube when in the open position. After the collet is in place and put inthe closed position, the septum 3021 is pushed over the needle andattached to the collet. In one embodiment, the septum includes aneedleless valve. In one embodiment, the needleless valve provides anairtight seal. The collet and septum together are referred to as aneedleless valve assembly. The collet prevents the septum from pushingthe coring tube forward during attachment of the septum. Once the septumis punctured by the coring tube and attached (e.g., by screwing) to thecollet, the syringe 3022 can be attached to the needless valve assembly.When the syringe plunger is withdrawn there is creation of a vacuumcondition within the coring tube. At this point, the entire assemblywith the coring tube may be retracted (FIGS. 3F and 3G), which causesthe cut tissue to be drawn into the syringe.

In one embodiment, a vacuum condition is applied at the same time as thecoring tube is withdrawn from the skin-related tissue structure, and theDMO is collected for example, into the syringe body. In anotherembodiment, the coring tube is withdrawn from the skin-related tissuestructure and then a vacuum condition is applied to the coring tuberesulting in recovery of the DMO, for example, into the syringe body. Inyet another embodiment, a vacuum condition is applied to the coring tubewhile the coring tube remains in the skin-related tissue structure andthe DMO is recovered, for example, into the syringe body. In stillanother embodiment, a drill is disconnected from a coring needle (1304)while holding a glued cap (1302) in place. (FIG. 13) Then a female luersyringe (1306) is attached directly to the clued cap. When the plungerof the syringe is withdrawn and the syringe/coring needle assemblyretracted from the skin-related tissue structure, the DMO is suctioneddirectly into the syringe body. Connection of the coring tube with thesyringe using a leurcap eliminates the need for a collet and needlelessvalve assembly.

In one embodiment, the syringe is partially filled with saline or othersuitable liquid, such that the tissue sample is withdrawn into a fluidenvironment which supports tissue viability.

The septum 3021 is needed to make sure that there is an air-tightconnection between the syringe 3022 and the interior of the coringneedle. If the syringe is attached to the collet directly without aseptum, withdrawal of the plunger of the syringe would not cause avacuum condition in the coring tube since a collet is in general notairtight. FIG. 4A (4012) shows one embodiment of a septum which may incertain embodiments be used in a method of harvesting a DMO of thisinvention.

In still another embodiment, recovery of a DMO is achieved bywithdrawing the cutting tool, from the skin-related structure, whereinthe DMO is retained within the cutting tool. The DMO 3024 may then berecovered from the cutting tool using positive pressure, e.g., theproximal end of the cutting tool may be attached to a syringe andpositive pressure applied by a syringe plunger so that a DMO is “pushed”from the distal end of the cutting tool. In addition, suitable fluids,such as sterile fluids, may be used to assist in removing the DMO fromcutting tool 3014.

In yet another embodiment, a DMO may be, for example, carefully removedfrom a cutting tool using mechanical means, such as tweezers or similartools, which can be used to grasp the distal end of the DMO located atthe distal end of the cutting tool.

As indicated at block 2016, the apparatus may then be removed from theharvest site. At this time, the outer sleeve may be removed manuallyfrom the skin-related tissue.

It will be appreciated by those skilled in the art that any combinationof the above actions may be implemented to perform harvesting accordingto embodiments of the invention. Further, other actions or series ofactions may be used.

Reference is also made to FIGS. 4C-E, which present exemplaryembodiments of a harvesting apparatus, wherein FIG. 4C presents anexample of a support structure attached to a vacuum source; FIG. 4Dpresents an example of an introducer, inner guide needle (4006) andouter sleeve (4008—white tube); FIG. 4E (4010) presents an example of acoring tube; and 4014 presents an example of a medical drill useful forrotating a cutting tool, and 4012 presents an example of a drill colletfor attaching the coring tube. FIGS. 4A-B present exemplary embodimentsof a syringe (FIG. 4A (4012)), septum (FIG. 4A 4004) and syringe collet(FIG. 4B) for use in the recovery of a dermal micro-organ.

According to some embodiments of the present invention, the abovedescribed manual procedures may be facilitated by an integratedapparatus (not shown) configured to perform some or all of the aboveprocedures for harvesting the DMO. For example, in regard to oneharvesting method embodiment, the integrated apparatus may be configuredto enable positioning and guiding the insertion of an introducer FIG.4D, guiding the insertion of coring tube FIG. 4E 4010 and controllingits rotational and forward movement during the cutting process, and/orremoving DMO from a coring tube. Such an apparatus may enable relativelysimple operation when performing a harvesting procedure.

Reference is now made to FIG. 5A, which schematically illustrates adermal to harvesting apparatus 5000 according to another exemplaryembodiment of the invention. As used herein, the term “supportstructure” refers to the body of the apparatus used to support askin-related tissue structure. The term “support structure” may also bereferred to herein as an “apparatus”. In this context an “apparatus” hasall the qualities and properties of a “support structure”.

Apparatus 5000 may include a guide channel 5003 and a vacuum chamber5001 including an elevated protrusion 5006. Elevated protrusion 5006 mayhave a predetermined size and/or shape adapted, for example, to enablethe creation of a “plateau” of a single layer of skin tissue in agenerally flat orientation, elevated above the trajectory of a coringtube 5016. For example, section 5006 may be higher than other sectionsof chamber 5001, such that a fat layer 5018 may be drawn into section5006 and supported along the trajectory of coring tube 5016. As aresult, after harvesting a DMO of a predetermined length, coring tube5016 may be advanced into fat layer 5018, thus separating the harvestedDMO from tissue surrounding the DMO. The harvested DMO may remain withincoring tube 5016 as it is withdrawn from the body, or a vacuum conditionmay be applied to the proximal end of the coring tube to suction the DMOfrom the coring tube. The configuration of Apparatus 5000 may eliminatethe need for forming an “exit” incision in the skin, thus enabling theharvesting of a DMO with only a single incision.

According to some exemplary embodiments of the invention, apparatus 5000may also include a drill stopper 5008 to enable manually advancingcoring tube 5016 for a predetermined distance along chamber 5001, e.g.,to a position in which coring tube 5016 has advanced into fat tissue5018.

According to some exemplary embodiment of the invention, apparatus 5000may also include a vacuum conduit 5004 which is connected to the vacuumchamber 5001 and a vacuum source 5002.

Reference is now made to FIG. 5B, which schematically illustrates across-sectional side view of a harvesting apparatus 5050 beingimplemented for externally supporting a skin-related tissue structure ata desired position according to another exemplary embodiment of theinvention.

In some embodiments, apparatus 5050 may include two channels 5064located at least partially along two sides of chamber 5001,respectively, to allow clamping epidermis layer 5020. Channels 5064 maybe positioned, e.g., centered, at a desired height, for example, atapproximately the same height as where the center of the DMO is to beharvested. In one embodiment, the central channel may be positioned at aheight of about 2 mm below upper surface of the vacuum chamber. so thatthe clamping may stabilize and/or support the tissue being cut.According to these exemplary embodiments, apparatus 5050 may alsoinclude two flexible membrane elements 5058, on either the inner surfaceor outer surface of channels 5064, so as to allow external clamping ofthe tissue without substantially affecting the vacuum condition appliedto chamber 5001. According to other embodiments of the invention,apparatus 5050 may not include elements 5058 and/or channels 5064.

According to the exemplary embodiment of FIG. 5B, improved stabilizationof dermis 5014 and/or improved prevention of recruitment of fat 5018into vacuum chamber 5001 may be accomplished by external clamping of theskin-related tissue structure supported within the vacuum chamber. Forexample, a clamping tool 5070, may be implemented to “pinch” theskin-related tissue structure supported inside vacuum chamber 5001,e.g., symmetrically. Two clamping ends 5054 of clamping tool 5070 may beinserted into channels 5056, respectively. Tool 5070 may be closed suchthat clamping ends 5054 may press down against flexible elements 5058.Thus, the skin-related tissue structure in chamber 5001 may be clampedfrom the sides without substantially affecting the vacuum condition inchamber 5001. A clamping force applied by clamping ends 5054 maycorrespond, for example, to a constant or variable force of a spring5062 or other suitable device.

Reference is now made to FIG. 6 and FIG. 11, which schematicallyillustrate embodiments of harvesting apparatuses according to someexemplary embodiment of the invention.

Apparatus 6000 may include a guide channel 6003 (FIG. 11, 1108) and avacuum chamber 6001 (FIG. 11, 2030) including two elevated protrusions6007 (proximal) and 6006 (distal) (FIG. 11, 1107 and 1109,respectively), and a central channel between the two protrusions. Theapparatus 6000 may further include one or more vacuum channels and avacuum conduit 6004 (FIG. 11, 1126) to fluidically connect the vacuumchamber with at least one vacuum source 6002. Elevated protrusions 6006and 6007 may have a predetermined size and/or shape adapted, forexample, to enable the creation of a “plateau” of a single layer of skintissue in a generally flat orientation, elevated above the trajectory ofa coring tube 6016. Elevated protrusions 6006 and 6007 may or may nothave the same size and shape. For example, section 6006 and 6007 may behigher than other sections of chamber 6001, such that epidermal 6020,dermal 6060 and fat layers 6018 may be drawn into sections 6006 and6007, respectively, such that in some embodiments, dermal tissue layers6060 are supported within the central channel within the trajectory ofthe coring tube 6016.

Application of a vacuum condition to an apparatus including a vacuumchamber including two elevated protrusions, for example, creates aprecise geometry of the skin-related tissue structure so that dermaltissue is harvested and a complete epidermal skin layer is notharvested. The presence of the proximal elevated protrusion incombination with use of the introducer avoids the harvesting of a plugof epidermal tissue at the proximal end of the DMO. After harvesting aDMO of a predetermined length, a coring tube 6016 may be advanced into afat layer 6018, thus allowing separation of the harvested DMO fromtissue surrounding the DMO. The harvested DMO may remain within coringtube 6016 as it is withdrawn from the body, or a vacuum condition may beapplied to the proximal end of the coring tube to suction the DMO fromthe coring tube.

The configuration of Apparatus 60000 may eliminate the need for formingan “exit” incision in the skin, thus enabling the harvesting of a DMOwith only a single puncture site.

According to some embodiments of the invention, the internal width of avacuum chamber of apparatus 5000 and/or 6000 is about 3.5 mm. In oneembodiment, a central channel may have a width of, for example, about 4mm. In another embodiment, a central channel may have a width of, forexample, 3.0 mm. Furthermore, in some embodiments, a central channel mayhave a height, excepting of the protrusions, of, for example, about 5mm. In other embodiments, other ranges, such as for example, 3-25 mm,may also be used for the width and/or height of central channel, forexample, any desired dimensions in the range of 3-25 mm may be used insome embodiments of the invention. The length of central channel may begenerally similar to the length of the DMO being harvested, for example,approximately 30 mm in length; however, other ranges, for example, inthe range of 5-100 mm. In another embodiment, the dimensions of thechannel length may be, for example, about 10-60 mm in length. In anotherembodiment, the dimensions of the channel length may be, for example,about 20-60 mm in length. In another embodiment, the dimensions of thechannel length may be, for example, about 20-50 mm in length. In anotherembodiment, the dimensions of the channel length may be, for example,about 20-40 mm in length. In another embodiment, the dimensions of thechannel length may be, for example, about 20-100 mm in length. Inanother embodiment, the dimensions of the channel length may be, forexample, about 30-100 mm in length. In another embodiment, thedimensions of the channel length may be, for example, about 40-100 mm inlength. In another embodiment, the dimensions of channel length may be,for example, about 50-100 mm in length. In another embodiment, thedimensions of the channel length may be, for example, about 60-100 mm inlength. In another embodiment, the dimensions of the channel length maybe, for example, about 70-100 mm in length. In another embodiment, thedimensions of the channel length may be, for example, about 80-100 mm inlength. In another embodiment, the dimensions of the channel length maybe, for example, about 90-100 mm in length. In another embodiment thelength may be around 20 mm. In another embodiment, the length may beabout 30 mm. In another embodiment, the length may be about 40 mm.

Prior to actual harvesting of the DMO, an apparatus of 6000 may be usedin conjunction with an introducer that includes, for instance, an innerneedle and an outer sleeve (FIG. 4D, 4008) for puncturing the skin andinserting a portion of an outer sleeve through the skin at the puncturesite and into fat tissue to prevent harvesting of epidermal tissue. Suchan introducer may be inserted through a guide channel 6003 of apparatus6000, wherein the tip of the outer sleeve reaches the area of proximalelevated protrusion 6006 and the tip of the needle extends beyond thedistal end of the outer sleeve. In one embodiment, the tip of the outersleeve passes through the skin layers and into the fat layer. In oneembodiment, the inner needle is a 14 GA needle. In another embodiment,the inner needle is a 12 GA needle. In another embodiment, the innerneedle is a 13 GA needle. In another embodiment, the inner needle is a15 GA needle. In yet another embodiment, the inner needle is a 16 GAneedle. In still another embodiment, the inner needle is a 17 GA needle.In another embodiment, the inner needle is an 18 GA needle. In anotherembodiment, the inner needle is a 19 GA needle.

As a result of withdrawing the inner needle, the outer sleeve may resideand extend from within the guide channel 6003 into dermal tissue 6060.Alternatively, the outer sleeve may reside and extend from within theguide channel 6003 into fat tissue 6018. In one embodiment, an outersleeve may be located from approximately the site of insertion toapproximately the center of the proximal elevated protrusion 6007. Theouter sleeve may act as a “sleeve” through which the coring needle maybe introduced directly into the fat and immediately in front of thedermal tissue which is to be harvested. The outer sleeve therebyprevents harvesting of epidermal tissue. In another embodiment, theouter sleeve may act as a “sleeve” through which the coring needle maybe introduced directly into the dermis, which is to be harvested. Inaddition, the outer sleeve protects the puncture site from the rotationand forward movement of the coring needle to prevent additional traumato the puncture site. In certain instances, the outer sleeve may have alow coefficient of friction to prevent resistance to the rotational andforward motion of the coring needle, thereby preventing unwanted heatgeneration.

According to some exemplary embodiments of the invention, apparatus 6000may also include a drill collet 6008 or glued cap to act as a hard stopenabling manual advancing of a coring tube 6016 for a predetermineddistance along chamber 6001, e.g., to a position in which coring tube6016 has advanced into fat tissue 6018 within distal elevated protrusion6006.

Tool 6016 may be connected to a motor, e.g., as described above, torotate tool 6016 at a rotational speed appropriate for cutting of thedermal tissue, for example, a relatively high rotational speed, forexample, a speed higher than 1,000 RPM, e.g., between 1,000 RPM and10,000 RPM. For example, tool 6016 may be rotated at a rotational speedhigher than 2,000 RPM, e.g., approximately 7,000 RPM. When complete, theforward and rotational movements of tool 6016 may be stopped, andcutting tool 6016 may be retracted with harvested DMO within it, therebyremoving the cutting tool from the harvest site. DMO may be removed fromcutting tool 6016, e.g., using a syringe to flush sterile fluid, forexample saline, through tool, or a vacuum source to draw out DMO from aback end (not shown) of cutting tool 6016, as described above.

It will be appreciated by those skilled in the art that apparatus 6000may enable harvesting of the DMO by forming only one incision orpuncture point. Furthermore, apparatus 6000 may be efficiently appliedfor harvesting a DMO from areas having relatively thick skin, e.g., froma region of the donor's back.

In some embodiments, elements of harvesting apparatus may be single useitems.

Reference is now made to FIGS. 10A and 10B, which show embodiments of asyringe with a septum and collet. FIG. 10A shows a syringe with a septumand collet attached to the back end of a cutting tool which is insertedthrough a support structure guide channel and an outer sleeve, whereinthe support structure is connected to a vacuum source. FIG. 10B shows anembodiment of a syringe with a collet and needless valve attached to theback end of a coring needle.

FIGS. 10A and 10B illustrate an embodiment of the invention, wherein afemale luer syringe is attached to the back end of the coring needle viaa collet and needleless valve. FIGS. 10A and 10B illustrate embodimentsthat may be used in order to suction the DMO out of the cutting tool andinto the syringe body.

It will be appreciated by those skilled in the art that the harvestingmethods and/or apparatuses according to embodiments of the invention,e.g., as described above, may include introducing thin tissue cuttingdevices within the dermis. Thus, the harvesting methods and/orapparatuses according to embodiments of the invention may enableharvesting the DMO with relatively minimal damage to the outer skinsurface, and therefore may provide a minimally invasive method ofharvesting the desired tissues.

Although some embodiments of the invention described herein may refer tomethods and/or apparatuses for harvesting a DMO, it will be appreciatedby those skilled in the art that according to other embodiments of theinvention at least some of the methods and/or apparatuses may beimplemented for any other procedures, e.g., plastic surgical procedures,dermatological procedures, or any other procedures including harvestingof tissues. For example, the methods and/or apparatuses according toembodiments of the invention may be implemented for harvesting dermaltissue to be used, e.g., in a subsequent implantation, as fillermaterial.

According to some embodiments of the present invention, a system andmethod are provided for ex-vivo (“in vitro”) handling or processing ofdermal micro-organs. In some embodiments, the dermal MOs may be directlyplaced into tissue culture wells or transduction chambers of abioreactor, for further processing. In some embodiments, e.g., if theDMO remains in the coring tube as it is withdrawn from the skin, the DMOmay be flushed out of the coring tube by the use of biologicallycompatible fluid, e.g., saline or growth medium, applied to the back endof the coring tube. The flushing of the DMO may be such that it isflushed directly into a chamber of a bioreactor. Alternatively, vacuummay be applied to a back end of the coring tube to “draw out” the DMO,e.g., directly into a chamber of a bioreactor.

II. Methods and Apparatuses for Implanting a DMO/DTMO

According to some embodiments of the present invention, a system andmethod are provided for implantation of DTMOs. After producing and/orprocessing of a DMO, for example, by genetically modifying the DMO, themodified DMO or DTMO may be implanted back into the patient, forexample, for protein or RNA based therapy. The number of full or partialDTMOs that are implanted may be determined by the desired therapeuticdosing of the secreted protein. DTMOs may be implanted subcutaneously orinto dermal tissue or at other locations within the body. Subcutaneousimplantation by use of an implantation needle, for example, may enablethe DTMO to remain in a linear form in a subcutaneous space. The linearform of implantation may help facilitate localization in case laterremoval or in-situ ablation of the DTMO is required, for example, inorder to stop treatment or reduce the dose of therapeutic protein. Otherknown geometrical implantation patterns could be used. The linearimplantation may also assist in the integration of the dermal tissue tothe surrounding tissue.

Reference is now made to FIGS. 7, 8A-E and 9A-E. FIG. 7 schematicallyillustrates a flowchart of a method of implanting a DMO/DTMO accordingto some exemplary embodiments of the invention, and to FIGS. 8A-E, whichpresent some embodiments of elements of an implanting apparatus. Themethods of implantation presented herein refer to implantation of eithera DMO or a DTMO, and the terms may be used interchangeable in describingthe methods and apparatus for implantation. For ease of reading only, aDTMO is recited in the description below wherein it is recognized thatthe term “DMO” is interchangeable with the term “DTMO” in the followingdescription.

As indicated at block 7002, a DTMO may be loaded into a syringe (FIG.8A). For instance a DTMO may be aspirated into a loading syringe.Loading may entail drawing up biologically compatible fluid, e.g.,saline or growth medium. Following loading of the DMTO in the syringe,an implanting tool, for example an implantation needle (FIG. 8B), may beattached to the syringe.

As indicated at block 7004, according to some exemplary embodiments ofthe invention, the DTMO, optionally together with surrounding sterilesaline fluid may be loaded into an implanting tool (FIG. 8B) byconnecting a loading syringe containing the DTMO with the proximal endof an implantation needle, and then gently loading the DTMO into theneedle using positive pressure. Alternatively, a DTMO may be aspirateddirectly into an implantation needle, for example, through the distalend of an implantation needle by withdrawing a plunger of a syringeattached to the proximal end of an implantation needle. Optionally, atip of the needle may have a removable short extension of silicontubing, or the like, affixed to it, to facilitate the aspiration of theDTMO into the needle cannula through the distal end while retracting theplunger of the syringe.

An implantation needle may have any suitable diameter, for example,between 17 GA and 8 GA. In one embodiment, the diameter of an implantingtool is about that of a 10 GA needle. In some embodiments, the tip of animplanting tool has a beveled edge. In other embodiments, the tip of animplanting tool does not have a beveled edge.

In some embodiments, after loading the DTMO into the implantationneedle, the proximal (back) end of the implanting needle is plugged toprevent the DTMO from coming out the back end of the tube. In otherembodiments, an adjustment of positive/negative pressure using theplunger of the loading syringe is used to keep the DTMO within theimplanting needle.

As indicated at block 7006 a local anesthetic may be optionallyadministered, e.g., as is known in the art, to the vicinity of anintended implantation site.

As indicated at block 7008, the method may further include positioningan apparatus including a support structure (FIG. 8D), at a givenimplantation site so that the support structure is in contact with anepidermal surface of the subject. In some embodiments, contact between asupport structure of this invention and the epidermal surface of asubject must be air-tight so that a vacuum seal may be formed at a laterstep. In one embodiment, an implantation site is on a subject's abdomen.In another embodiment, an implantation site is on a subject's back.Alternatively, the implantation site may be at another location on thesubject's body.

In some instances, dosage may be adjusted based on thenumber/size/efficacy of DTMOs to be implanted. For example, multipleDTMOs may be implanted consecutively during a single procedural timeperiod in order to reach a target dose. In one embodiment, an implantmarker template may be used prior to positioning an implanting apparatuson a subject's epidermal surface (step 7008), in order to mark multiplesites for implanting. In one embodiment, an implanting marker templateis positioned on the epidermal surface of a subject, and the epidermalsurface is then marked to indicate, for example, anesthesia lines andalignment lines for later placement of the support structure 9006. Inone embodiment, the surface is marked using a surgical pen or marker. Inone embodiment, the surface is marked using a non-permanent dye or ink.

As indicated at block 7010, a support structure FIG. 8D, which mayinclude a vacuum chamber with at least one elevated protrusion and guidechannel 9008, wherein the elevated protrusion, 9007, is adjacent to theguide channel. For instance a support structure of FIG. 5A or 5B may beused to hold and support the skin-related tissue structure in place forproper implanting of a DTMO, for example, to create a unique geometry ofthe skin, such that the path of implantation is precisely controlled.For example, application of vacuum conditions causes a vacuum to beformed within the vacuum chamber thereby drawing the epidermal surfaceof the skin-related structure into the interior of the supportstructure, wherein a central channel may support epidermal and dermalskin layers, and possibly fat layers, of the skin-related structure. Inexemplary embodiments, a vacuum condition may cause the skin-relatedstructure to be held at an inner support surface of the vacuum chamber.The guide channel may provide guidance and/or stability for animplanting tool to ensure proper implanting along a linear implantingaxis, which in one embodiment is in the subcutaneous space, such asbetween the dermis and fat layers. In some embodiments, implantingresults in a DTMO remaining substantially linear after implantation. Insome embodiments, the implanting axis is coaxial with the needle guidechannel and central channel. In another embodiment, a DTMO is implantingwithout ensuring linearity.

As indicated at block 7012 and FIGS. 9A and 9B, and similar to the usefor methods of harvesting described above, an introducer, FIG. 8C, maythen be used to puncture the skin-related tissue by inserting theintroducer through the guide channel of the support structure and intothe skin-related structure along the implanting axis. This singlepuncture site may be used for all further entry into the skin-relatedstructure. In this way damage and scarring to the subject is limited. Inaddition, use of an introducer eliminates exposure of the loaded DTMOwithin an implantation tool to a vacuum condition while implantationtool is penetrating the skin-related structure. Exposure of the DTMOloaded in the implantation needle to vacuum conditions present withinthe support structure may lead to a risk of the DTMO being suctionedinto the vacuum line.

An introducer composed of an inner needle 9010 and an outer sleeve 9012,fitted as a sheath over the inner needle, may be inserted into theskin-related structure such that the distal edge of the outer sleeveextends into the region of tissue just under the elevated protrusion.Under vacuum conditions, a precise geometry of the skin-related tissuestructure is created such that insertion of the introducer is forexample, generally perpendicular to the skin surface at the point ofpenetration. In one embodiment, the inner needle of the introducer andthe implanting tool have about the same dimensions of diameter. Forinstance, the diameter of an inner needle and of an implanting tool mayeach be about those of a 10 GA needle.

As indicated at block 7014 and FIG. 9B, the inner needle is withdrawnfrom the skin-related tissue structure while the outer sleeve component9012 of the introducer is positioned and remains in place. The result ofthis action is that the outer sleeve is positioned coaxially with theimplanting axis and extends into the skin-related tissue structure. Inpreferred embodiments, the distal edge of the outer sleeve extends intothe fat under the elevated protrusion. In other preferred embodiment,the distal edge of the outer sleeve extends into dermis under theelevated protrusion.

The outer sleeve may include a thin needle, tube or any other suitablethin, generally straight, object able to be placed inside the dermis orin a subcutaneous space. For example, an outer sleeve may include aneedle of size 6-18 GA, for example, about 10 GA or 14 GA, as is knownin the art. Alternatively, an outer sleeve may include a plastic tube.In one embodiment, an outer sleeve includes high-density polyethylene(HDPE) tubing. In another embodiment, an outer sleeve includes Teflon®.In yet another embodiment, an outer sleeve includespolytetrafluoroethylene (PTFE) tubing. In still another embodiment, anouter sleeve includes fluorinated ethylene propylene (PEP) tubing.

An introducer may be inserted into the dermis or subcutaneous space bybeing pushed generally perpendicular to the skin surface at the point ofpenetration. In one embodiment, an inner needle is beveled. In such acircumstance, introduction of the inner needle part of an introducer maybe with the bevel side facing down. In another embodiment, the bevelside is facing upward. In yet another embodiment, the bevel side isfacing any direction in between upward and downward. In still anotherembodiment, an inner needle is not beveled.

As indicated at block 7016 and FIG. 9C, an implanting tool 9014 with theloaded DTMO 9016 may be inserted through the support structure guidechannel and the outer sleeve, and advanced along an implanting axis intothe desired location, e.g., in the subcutaneous space, within a fatlayer, at the interface of fat layer and dermis layer, or into dermaltissue layer, along a distance approximately equivalent to the length ofthe DTMO. A silicon tubing extension, if used for loading of the DTMO1716, would be removed prior to insertion of the implanting tool throughthe needle guide channel. An implanting tool may be a needle, forinstance 6 GA-14 GA. In one embodiment, an implanting tool may be a 10GA needle. In another embodiment, an implanting tool may be a 14 GAneedle. In one embodiment, the implanting tool is inserted with thebevel up. In another embodiment, the implanting tool is inserted withthe bevel down. Once the implanting tool has been advanced for placementof a DTMO at a desired location, the plug at the proximal end of theimplanting tool, if used, is removed.

As indicated at block 7018 and FIG. 9D, a stopper tool including astopper tool body 9018 and a stopper pin 9020 (FIG. 8E) may be connectedwith an apparatus such that it is secured relative to the apparatus anda stopper pin inserts within the implanting needle 9016 through theproximal end.

As indicated at block 7020 and FIG. 9E, the implantation needle may beretracted through e.g., the subcutaneous space, releasing the DTMO fromthe implantation needle and laying the DTMO linearly along the needletract. In one embodiment, in order to ensure linear placement of a DTMO,a stopper pin, 9020, of a stopper tool (FIG. 8E) is inserted within theproximal end of the implanting needle. In another embodiment, assistancemay be given to help release the DTMO for example by connecting asyringe to the proximal end of the implantation needle and gentlyproviding positive pressure with the syringe plunger, possibly duringretraction of the implantation needle.

In exemplary embodiments of the invention, a stopper of FIG. 8E isattached to the support structure FIG. 8D, such that the rod of thestopper is internal and coaxial with the implanting tool. In oneembodiment, the stopper's association with the support structure is suchthat its placement is set, e.g., locked-in-place, relative to otherelements of the implanting apparatus. For instance, the stopper rod mayfit inside the back end of the implanting needle such that the rod isbrought into close proximity with the loaded DTMO within the implantingneedle, and the implanting needle may be withdrawn over the stationaryrod of the stopper. Retraction of the implanting needle over the rodmay, in one embodiment, be for the full extent of the rod. In anotherembodiment, retraction may be over a part of the rods length. As the rodis stationary, retraction of the needle may result in the rod extendingbeyond the beveled tip of the implanting needle after retraction.Retraction of the implanting needle over the rod of the stopper may insome instances prevent the DTMO from being pulled back together with theimplanting needle. In yet another embodiment, retraction may result inthe DTMO being released into the target site, e.g. subcutaneous space,in a linear form.

Linear implantation of a DTMO may provide better exposure of theimplanted tissue to surrounding environment. For example, linearimplantation may facilitate better integration of the DTMO. In addition,linear implantation may facilitate diffusion of a secreted recombinantproduct, e.g., a recombinant protein or portion thereof. Moreover,linear implantation may facilitate increased angiogenesis in the regionof the DTMO. If, at a future date, it is required that a DTMO be excisedor ablated, linear positioning provides a known orientation and locationof a given DTMO. In one embodiment, implantation results in a DTMO beingplaced linearly within a subcutaneous space. In another embodiment,implantation results in a DTMO being placed linearly within a tissue ofthe same kind as the DTMO, e.g., dermal tissue. In yet anotherembodiment, implantation results in a DTMO being placed linearly deeperin the body.

As indicated at block 7022, the apparatus may then be removed from theimplant site. In addition, the outer sleeve may be removed at this time.

It will be appreciated by those skilled in the art that any combinationof the above actions may be implemented to perform implanting accordingto embodiments of the invention. Further, other actions or series ofactions may be used.

In addition and without repeating the description and all of theembodiments of apparatuses 5000 and 6000 (FIGS. 3-7 and FIG. 11)described above as apparatuses for harvesting, e.g., support structuresfor harvesting a DMO, in some embodiments of the invention apparatuses5000 and 6000 may also be used in methods of implanting a DTMO.Accordingly, in some embodiments of the invention, apparatuses 5000 or6000 may also be used in methods of implanting.

III. Methods and Apparatuses for Excising a DMO/DTMO

According to some embodiments of the present invention, a system andmethod are provided for in-vivo demarcation and localization of theimplanted dermal micro-organs. Identification of the location of asubcutaneous implantation or implantation at any other location in thebody, of processed tissue, such as a DTMO, may be important, forexample, in the case where it is necessary to stop the proteintreatment, or to decrease the dosage of the secreted protein. Forexample, termination or titration of dosage may be performed by removingone or more DTMOs entirely and/or by ablating one, a portion of one, ormore than one of the implanted DTMOs. In order to identify asubcutaneously implanted DTMO, according to one embodiment, the DTMO maybe colored prior to implantation by an inert, biocompatible ink or staincontaining, for example, a chromophore, which may be visible to thenaked eye or may require special illumination conditions to visualizeit. In this way a DTMO may be distinguished from its surrounding tissueby visual inspection and/or by use of enhanced imaging means.

According to one embodiment, at least the peripheral surface of a DTMOmay be coated with, for example, biocompatible carbon particles,biocompatible tattoo ink, or other suitable materials including titaniumparticles, magnetic particles and/or microspheres. Once implantedsubcutaneously, the DTMO may be visible with the naked eye, by suitableenhanced imaging device, or other means of detection. Other ways toenhance the visibility of an implanted DTMO may include using a stronglight source above the skin surface, or pinching the skin and directingthe light source at the skin from one side, such that the skin mayappear translucent and the dyed DTMO may be more visible. Alternatively,the stain may be fluorescent, visible only when illuminated using UVlight, such as using fluorescent plastic beads.

According to another embodiment, the location of a subcutaneouslyimplanted DTMO may be identified by co-implanting a biocompatiblestructure along with the DTMO. An example of such a biocompatiblestructure is a non-absorbable single stranded nylon suture commonly usedin many surgical procedures. Such a suture may be implanted in the sameimplantation tract with the DTMO, or may be implanted directly above theDTMO in the upper dermis or below the DTMO in the fat, such that thespatial location of the DTMO may be determined by the suture location.Further, the depth of the DTMO may be known to be at the depth of thesubcutaneous space. The suture may be visible to the naked eye, observedwith the assistance of illumination means, and/or observed with the aidof other suitable imaging means, such as ultrasound. Alternatively, thesuture can be fluorescent, and visible through the skin underappropriate UV illumination. The suture may alternatively be of anabsorbable material, so that it may enable determination of localizationfor a desired period of time, such as a few months.

According to another embodiment, the DTMO may be genetically modified orengineered to include a gene to express a fluorescent marker or othermarker capable of being visualized. For example, the DTMO can bemodified with the GFP (Green Fluorescent Protein) gene or Luciferasereported gene, which, for example, may be expressed along with the genefor the therapeutic protein. In this manner, the DTMO may be visualizednon-invasively using appropriate UV or other suitable illumination andimaging conditions.

According to another embodiment, one or more tattoo marks, e.g. smalltattoo dots, can be applied on the skin in the vicinity of theimplantation site. In a preferred embodiment, a small tattoo dot isapplied to the skin at either end of a linearly implanted DTMO. Thetattoo ink can be permanent, or temporary, such as ink used for cosmeticmake-up use.

According to some embodiments of the present invention, a system andmethod are provided for removal or ablation of implanted DTMOs. In acase, for example, where DTMO-based therapy to a patient must beterminated, or if the protein secretion must be decreased, one or moreimplanted DTMO may be partially or entirely removed, or partially orentirely ablated. In one embodiment, the DTMO may be surgically removed.

According to one embodiment in which small tattoo dots are applied onthe skin at either end of a linearly implanted DTMO, surgical excisionof the DTMO can be accomplished by resection of an elliptical tissuesample, including at least both tattoo dots and including all of thelayers of skin and some subcutaneous tissue to ensure that the DTMO hasbeen removed. The excision site can then be sutured closed.

In addition and without repeating the description and all of theembodiments of apparatuses 6000 and 1100 (FIGS. 3-6 and 11) describedabove as apparatuses for harvesting, e.g., support structures forharvesting a DMO, in some embodiments of the invention apparatuses 6000and 1100 may also be used in methods of excising a DTMO.

Reference is now made to FIG. 12, which illustrates an exemplaryembodiment of excising a DTMO. It will be appreciated by those skilledin the art that any combination of the above actions for harvesting maybe implemented to perform excising of an DTMO according to embodimentsof the invention. Further, other actions or series of actions may beused.

Without repeating all of the embodiments for harvesting described indetail above, reference is now made to FIG. 12. Briefly, at block 1202the location of the implanted subcutaneous DTMO may be determined. Atblock 1203, a local anesthetic may be optionally administered at thesite of DTMO removal. At block 1204 a support structure may bepositioned over the site of the DTMO to be removed. A support structure(e.g., FIG. 4C; FIGS. 5A-B, FIG. 6, FIG. 11), which may include a vacuumchamber and a guide channel, may be used to hold and support theskin-related tissue structure for proper excising of a DTMO and minimalsurrounding tissue thereof.

At block 1206, vacuum conditions are applied and a skin-related tissuestructure including the DTMO to be excised may be shaped so that thetissue containing the DTMO to be excised is within a central channel andis aligned with a cutting axis.

At block 1208, an introducer including an inner needle and an outersleeve may be used to puncture the skin-related tissue by inserting theintroducer through the guide channel of the support structure and intothe skin-related tissue structure at a point of penetration. The innerneedle is then removed and the outer sleeve remains positioned with thedistal end of the outer sleeve residing in a region proximal to the DTMOto be excised.

At block 1210, a core of tissue that includes the DTMO may be harvested.A coring needle, of the same or larger diameter than that of the DMOharvesting needle (for example, 11 GA or similar), may be insertedthrough the guide channel and outer sleeve along a cutting axis in orderto harvest the previously implanted DTMO. In one embodiment, a DTMO isexcised using a coring tube similar to, or larger in diameter than thatused for direct harvesting of the DMO. In one embodiment, additionaltissue surrounding the DTMO being excised is harvested during theexcision of the DTMO. In one embodiment, the additional tissue includesepidermal tissue. In one embodiment, the additional tissue includesdermal tissue not associated with the DTMO. In one embodiment, theadditional tissue includes fat tissue. In one embodiment, such a coringapproach may be combined with vacuum suction at the proximal end of thecutting tool to help remove the cut tissue sample from the body.

According to an embodiment of the present invention, minimally invasiveor non-invasive methods of ablating the DTMO in-situ may be used to makethe procedure less traumatic and less invasive for the patient. In oneembodiment, potentially in conjunction with the case of the dyed DTMO, alaser, for example, a non-invasive Yag laser may be used. The energy ofthe Yag laser, for example, may be selectively absorbed by thechromophore of a dyed DTMO, such that the energy is primarily directedto the DTMO, with minimum damage caused to the surrounding tissue. Otherlight energy sources may also be used. Alternatively, such a laserapproach can be used with other means of locating the DTMO other thanuse of a dye.

According to another embodiment, the DTMO may be ablated by deliveringdestructive energy from a minimally invasive probe inserted into thesubcutaneous space along the length of the DTMO. Such a probe may enabledelivery of a variety of energy types, including radio frequency,cryogenic temperatures, microwave, resistive heat, etc. A co-implantedstructure, such as a suture, may be used to determine the location ofthe DTMO, thereby enabling the probe to be inserted subcutaneously, forexample, along or directly above or below the suture. In such a case,for example, the destructive energy may be delivered while the suture isstill in place. Alternatively, the suture may be removed after placementof the probe and before delivery of the destructive energy. The amountof energy applied may be either that required to denature the proteinsin the tissue such as during coagulation by diathermy. Additionally oralternatively, the amount of energy applied may be as much as is used inelectro-surgical cutting devices, which char tissue. Of course, othermeans of localization and other means of delivering destructive energymay be used.

IV. Methods and Apparatuses for Processing a DMO

After a DMO is harvested, e.g., according to embodiments of the presentinvention, the DMO is optionally genetically altered. Methods andApparatuses for processing a DMO have been described in detail in UnitedStates Publication No. US-2012/0201793-A1, which is incorporated hereinby reference in full.

In one embodiment, the invention provides a method of delivering a geneproduct of interest into a subject by implanting the geneticallymodified DMO of the invention into a subject.

The invention contemplates, in one aspect, the use of the geneticallymodified DTMO for transplantation in an organism. As used herein theterms “administering”, “introducing”, “implanting” and “transplanting”may be used interchangeably and refer to the placement of the DTMO ofthe invention into a subject, e.g., an autologous, allogeneic orxenogeneic subject, by a method or route which results in localizationof the DTMO at a desired site. The DTMO is implanted at a desiredlocation in the subject in such a way that at least a portion of thecells of the DTMO remain viable. In one embodiment of this invention, atleast about 5%, in another embodiment of this invention, at least about10%, in another embodiment of this invention, at least about 20%, inanother embodiment of this invention, at least about 30%, in anotherembodiment of this invention, at least about 40%, and in anotherembodiment of this invention, at least about 50% or more of the cellsremain viable after administration to a subject. The period of viabilityof the cells after administration to a subject can be as short as a fewhours, e.g., twenty-four hours, to a few days, to as long as a few weeksto months or years.

Alternatively, the DTMO, which includes genetically modified cells canbe kept in vitro and the therapeutic agent, left in the supernatantmedium surrounding the tissue sample, can be isolated and injected orapplied to the same or a different subject.

Alternatively or additionally, a DTMO can be cryogenically preserved bymethods known in the art, for example, without limitation, gradualfreezing (0° C., −20° C., −80° C., −196° C.) in DMEM containing 10%DMSO, immediately after being formed from the tissue sample or aftergenetic alteration.

In accordance with an aspect of some embodiments of the invention, thenumber of DTMOs to be implanted is determined from one or more of:Corresponding amounts of the therapeutic agent of interest routinelyadministered to such subjects based on regulatory guidelines, specificclinical protocols or population statistics for similar subjects.Corresponding amounts of the therapeutic agent such as protein ofinterest specifically to that same subject in the case that he/she hasreceived it via injections or other routes previously. Subject data suchas weight, age, physical condition, clinical status. Pharmacokineticdata from previous tissue sample which includes a genetically modifiedcell administration to other similar subjects. Response to previous DTMOadministration to that subject.

In accordance with an aspect of some embodiments of the invention, onlysome of the DTMOs are used in a given treatment session. The remainingDTMOs may be returned to maintenance (or stored cryogenically orotherwise), for later use.

There is also provided in accordance with an embodiment of theinvention, method of adjusting the dosage of a therapeutic agentproduced by a DTMO implanted in a subject and excreting a therapeuticagent, including (a) monitoring level of therapeutic agent in thesubject; (b) comparing the level of agent to a desired level; (c) if thelevel is lower than a minimum level, then implanting additional DTMO;(d) and if the level is higher than a maximum level, then ablating orremoving one or more of the implanted DTMOs. Optionally, the methodincludes periodically repeating (a)-(d). Alternatively or additionally,ablating or removing consists of ablating or removing a portion of oneor more of the implanted DTMOs. Optionally, removing includes surgicalremoval. Optionally, ablating includes killing a portion of theimplanted DTMO.

As described above with reference to FIG. 1, at least part of theprocess of sustaining the DMO during the genetic alteration, as well asthe genetic alteration itself, may be performed in a bioreactor.

EXAMPLES Example 1 Harvesting of A Dermal Micro-Organ

Dermal micro-organs were harvested from a human subject under sterileconditions.

Experimental Procedure

With the subject prone, a harvest site on the lower abdomen wasselected, disinfected, marked with guidelines, and injected with localanesthesia. The harvest site was in an area of healthy skin, free ofstretch marks or other obvious skin abnormalities. A sterile harvestingsupport (FIG. 4C) structure containing a vacuum control hole wasconnected to a vacuum source, the vacuum turned on and the supportstructure placed on the subject's epidermis at the selected harvest sitewith the vacuum control hole uncovered.

A finger was placed over the vacuum control hole causing a vacuum toraise the skin-related tissue structure into the vacuum chamber.

With the sharpened bevel point of the Introducer inner needle facingdown (FIG. 4D, 4006), the Introducer (4006 and 4008) was inserted intothe needle guide of the support structure, quickly and to the full stop.The Introducer's inner needle 4006 was then removed, leaving behind theIntroducer sleeve 4008.

Next the sharpened tip of the coring needle attached to the medicaldrill was inserted into the Introducer sleeve and gently pushed forwarduntil the tip reached the distal end of the sleeve. The drill was thenactivated and pushed forward to the full stop, pushing the tip of thecoring needle through the dermal tissue and into fat tissue. At thispoint the vacuum was deactivated by removing the finger from the vacuumcontrol hole.

The drill was then disconnected from the coring needle and theneedleless valve syringe assembly was connected to the coring needle byslipping the collet onto the needle and tightening it onto the needle,and then by piercing the septum with the exposed end of the coringneedle and connecting that to the collet. The syringe was connected tothe septum and the plunger of the syringe was withdrawn to create avacuum, while the syringe was retracted together with the coring needle.The DMO was suctioned into the syringe body during this withdrawalprocess (FIG. 3G).

Experimental Results

Multiple DMOs were harvested. An isolated harvested DMO is shown in FIG.14A in comparison to a toothpick, wherein the DMO is approximately 30 mmin length. As shown in FIG. 14B (1402), there was only minimal scarringof the skin tissue at the harvest sites.

Example 2 Implanting of A Dermal Micro-Organ

Dermal micro-organs were implanted into a human subject under sterileconditions.

Experimental Procedure

Similar to preparations for harvesting, the implantation of dermalmicro-organs into a human subject began with the subject prone, withimplant sites selected on the lower abdomen, disinfected, marked withguidelines to align the support structure for implanting, and the sitesinjected with local anesthesia. A sterile implanting support structure(FIG. 8D) containing a vacuum control hole was connected to a vacuumsource, the vacuum turned on and the support structure placed on thesubject's epidermis at the marked implantation sites with the vacuumcontrol hole uncovered.

A finger was placed over the vacuum control hole causing a vacuum toraise the skin-related tissue structure into the vacuum chamber.

With the sharpened bevel point of the Introducer inner needle facingdown (FIG. 8C), the Introducer was inserted into the needle guide of thesupport structure, quickly and to the full stop. The Introducer's innerneedle was then removed, leaving behind the Introducer sleeve.

Next the implantation needle loaded with a DMO at the distal end wasinserted into the Introducer sleeve, and pushed forward to the fullstop. The stopper element was than connected to the implanting apparatusby inserting the stopper pin within the inner lumen of the implantationneedle. The stopper pin was moved forward and brought into closeproximity to loaded DMO within the implantation needle. The stopper bodywas affixed to the implanting support structure so that the stopper pinremains stationary while the implantation needle was retracted over thestopper pin and the DMO was linearly implanting within the subcutaneousspace at the implantation site.

The implanting tools were carefully removed from the implant site andthe vacuum was removed by removing the finger from the vacuum hole. Atattoo dot was made with semi-permanent ink on the surface of the skinat either end of the linearly implanted DMO to demark the location ofthe implantation site.

Experimental Results

Multiple DMOs were implanted. As shown in FIG. 14B, DMOs were implantedin the lower abdomen (1404), wherein tattoo dots identify their site ofimplantation. There was only minimal scarring of the skin tissue at theimplantation sites.

It will thus be clear, the present invention has been described usingnon-limiting detailed descriptions of embodiments thereof that areprovided by way of example and that are not intended to limit the scopeof the invention. For example, only a limited number of genetic changeshave been shown. However, based on the methodology described herein inwhich live tissue is replanted in the body of the patient, and theviability of that tissue in the body after implantation, it is clearthat virtually any genetic change in the tissue, induced by virtuallyany known method will result in secretions of target proteins or othertherapeutic agents in the patient.

Variations of embodiments of the invention, including combinations offeatures from the various embodiments will occur to persons of the art.The scope of the invention is thus limited only by the scope of theclaims. Furthermore, to avoid any question regarding the scope of theclaims, where the terms “comprise” “include,” or “have” and theirconjugates, are used in the claims, they mean “including but notnecessarily limited to”.

Further, as used herein, the term “comprising” is intended to mean thatthe system includes the recited elements, but not excluding others whichmay be optional. By the phrase “consisting essentially of” it is meant amethod that includes the recited elements but exclude other elementsthat may have an essential significant effect on the performance of themethod. “Consisting of” shall thus mean excluding more than traces ofother elements. Embodiments defined by each of these transition termsare within the scope of this invention.

Further, as used herein, the term “about”, refers to a deviance ofbetween 0.0001-5% from the indicated number or range of numbers. In oneembodiment, the term “about”, refers to a deviance of between 1-10% fromthe indicated number or range of numbers. In one embodiment, the term“about”, refers to a deviance of up to 25% from the indicated number orrange of numbers.

Further, as used herein, the term “a” or “one” or “an” refers to atleast one. In one embodiment the phrase “two or more” may be of anydenomination, which will suit a particular purpose.

Further, as used herein, the term “treatment” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or lessen a targeted pathologic condition or disorder.Thus, in one embodiment, treating may include directly affecting orcuring, suppressing, inhibiting, preventing, reducing the severity of,delaying the onset of, reducing symptoms associated with a disease,disorder or condition, or a combination thereof. Thus, in oneembodiment, “treating” refers inter alia to delaying progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. In one embodiment, “preventing”refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, or acombination thereof. In one embodiment, “suppressing” or “inhibiting”,refers inter alia to reducing the severity of symptoms, reducing theseverity of an acute episode, reducing the number of symptoms, reducingthe incidence of disease-related symptoms, reducing the latency ofsymptoms, ameliorating symptoms, reducing secondary symptoms, reducingsecondary infections, prolonging patient survival, or a combinationthereof.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. An apparatus for implanting a dermal micro-organinto a skin-related tissue structure, the apparatus comprising: a. aloading syringe comprising a first tubular element; b. an implantingtool comprising a second tubular element having a lumen; c. a supportstructure to hold the skin-related tissue structure in place; whereinthe support structure comprises i. a vacuum chamber comprising an innersupport surface able to hold the skin-related tissue structure in adesired shape and position to enable the implanting tool to implant thedermal micro-organ into the skin-related tissue structure, ii. one ormore vacuum channels to fluidically connect the vacuum chamber with atleast one vacuum source, and iii. a guide channel providing a site ofinsertion of the implanting tool into the support structure andproviding an implanting axis for implantation of the dermal micro-organinto the skin-related tissue structure; d. an introducer for puncturingsaid skin related tissue structure at the site of insertion of theimplanting tool and prior to insertion of the implanting tool, whereinthe introducer is able to be inserted through the guide channel; and e.a stopper tool able to be connected to said support structure, saidstopper tool comprising a stopper tool body and a rod, the rod able tobe inserted within the lumen of the second tubular element of theimplanting tool prior to retraction of the implanting tool from theskin-related tissue structure, said stopper tool assisting inmaintaining the position of the implanted dermal micro-organ.
 2. Theapparatus of claim 1, wherein said vacuum chamber further comprises: a.at least one elevated protrusion, said elevated protrusion able tosupport a plateau of epidermal and dermal skin layers from saidskin-related tissue structure above the implanting axis; and b. acentral channel co-serial with the guide channel and distal to saidelevated protrusion relative to said site of insertion, said centralchannel able to support skin-related tissue.
 3. The apparatus of claim1, wherein said introducer comprises: a. an inner tubular element, andb. an outer tubular element, wherein the inner tubular element insertsthrough the outer tubular element and extends beyond the distal end ofthe outer tubular element, the inner and outer tubular elements togetherinserting at said site of insertion coaxially within the guide channel;and the outer tubular element capable of remaining coaxial and withinthe guide channel upon withdrawal of the inner tubular element from theguide channel after puncturing of the skin-related tissue structure. 4.The apparatus of claim 3, wherein the inner tubular element is a needleand the outer tubular element is an outer sleeve.
 5. The apparatus ofclaim 1, wherein said second tubular element of the implanting toolcomprises a needle able to advance along the implanting axis and implanta dermal micro-organ along said implanting axis.
 6. The apparatus ofclaim 5, wherein the needle tip is beveled.
 7. The apparatus of claim 1,wherein the implanting axis is substantially linear and generallyparallel to the skin-related tissue structure surface.
 8. The apparatusof claim 1, wherein the implanting axis allows for implantation into afat layer or between a dermis and a fat layer.
 9. The apparatus of claim1, wherein the first tubular element is capable of connecting with oneend of the second tubular element to facilitate transfer of the dermalmicro-organ from the loading syringe to the implanting tool.
 10. Theapparatus of claim 2, wherein the elevated protrusion of the supportstructure allows the introducer to be pushed generally perpendicular tothe skin-related tissue structure surface at the point of insertionwhile the implanting axis is substantially linear and generally parallelto the skin-related tissue structure surface.
 11. The apparatus of claim2, wherein the elevated protrusion of the support structure allows theintroducer to penetrate the dermis or subcutaneous space.
 12. A methodof implanting a dermal micro-organ into a skin-related tissue structurecomprising the steps of: a. loading said dermal micro-organ into aloading syringe, said loading syringe comprising a first tubularelement; b. transferring said dermal micro-organ from said loadingsyringe into an implanting tool, said implanting tool comprising asecond tubular element; c. placing a support structure at animplantation site, wherein said support structure supports theskin-related tissue structure at the implantation site, and wherein thesupport structure comprises: i. a vacuum chamber comprising an innersupport surface able to hold the skin-related tissue structure in adesired shape and position to enable the implanting tool to implant thedermal micro-organ into the skin-related tissue structure, ii. one ormore vacuum channels to fluidically connect the vacuum chamber with atleast one vacuum source, and iii. a guide channel providing animplanting axis for implantation of the dermal micro-organ into theskin-related tissue structure; d. inserting an introducer through theguide channel and puncturing the skin-related tissue structure with theintroducer, and optionally withdrawing at least a portion of theintroducer following puncturing; e. advancing said implanting toolthrough the guide channel and into said skin-related tissue structurealong said implanting axis; f. providing a stopper comprising a stopperbody and rod, and inserting the rod into the lumen of the second tubularelement of the implanting tool; and g. withdrawing said second tubularelement and stopper rod, wherein said dermal micro-organ remains withinsaid skin-related tissue structure.
 13. The method of claim 12, whereinsaid dermal micro-organ is a genetically modified dermal micro-organ.14. The method of claim 12, wherein said vacuum chamber comprises atleast one elevated protrusion and a central channel, said elevatedprotrusion proximal and said central channel distal to said site ofinsertion; and wherein applying a vacuum condition aligns saidimplanting axis to be co-axial with said central channel.
 15. The methodof claim 12, wherein said support structure supports the skin-relatedtissue structure at the implantation site by applying a vacuum conditionto said vacuum chamber.
 16. The method of claim 12, wherein the methodcreates only a single puncture point in said skin-related tissuestructure.
 17. The method of claim 12, wherein the dermal micro-organ isimplanted in a substantially linear form.
 18. The method of claim 12,wherein the dermal micro-organ is implanted within a fat layer orbetween a dermis and a fat layer.
 19. The method of claim 12, whereinthe dermal micro-organ consists essentially of a plurality of dermalcomponents lacking a complete epidermal layer.
 20. The method of claim12, wherein the dermal micro-organ is transferred from the loadingsyringe to the implanting tool by connecting the first tubular elementto one end of the second tubular element.
 21. The method of claim 12,wherein said introducer comprises: a. an inner tubular element, and b.an outer tubular element, wherein the inner tubular element insertsthrough the outer tubular element and extends beyond the distal end ofthe outer tubular element, the inner and outer tubular elements togetherinserting at said site of insertion coaxially within the guide channel;and after puncturing the inner tubular element is withdrawn from theguide channel after puncturing of the skin-related tissue structurewhile the outer tubular element remains within the guide channel. 22.The method of claim 21, wherein the inner tubular element comprises aneedle.
 23. The method of claim 12, wherein the second tubular elementof the implanting tool comprises a needle.
 24. The method of claim 12,wherein withdrawing the second tubular element and stopper rod comprisesretracting the second tubular element over the rod, wherein said rodelement remains static relative to the support structure.